International Conference on Lasers, Applications and Technologies (LAT2016)

[1.01] Spectral luminescent properties of bacteriochlorin and aluminum phthalocyanine nanoparticles as hydroxyapatite implant surface coating

Alina S. Sharova¹, Yuliya S. Maklygina², Biswanath Kundu³, Vamsi K. Balla³, Rudolf Steiner⁴ and Victor B. Loschenov^{1, 2}

¹ National Research Nuclear University (NRNU), Moscow Engineering Physics Institute (MEPhI), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

² A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

³ Bioceramics and Coating Division, CSIR – Central Glass & Ceramic Research Institute, 196 Raja S.C. Mullick Road, Kolkata, 700 032, West Bengal, India

⁴ Institut für Lasertechnologien in der Medizin und Meßtechnik an der Universität Ulm, Helmholtzstr. 12, 89081 Ulm, Germany

E-mail contact: join93@rambler.ru

Abstract: Spectral luminescent properties of coating for hydroxyapatite implants were investigated experimentally. Crystalline bacteriochlorin and aluminum phthalocyanine nanoparticles with photobactericidal properties were employed as coating for the implants. This research opens the prospect of providing local inflammatory and autoimmune reactions prevention within the implantation area.

1 Introduction

Surgical intervention with subsequent implant placement is a difficult process in terms of post-operative recovery, preventing of inflammatory responses and a possible implant rejection process. Currently, the most promising methods to induce a bactericidal effect in the area of the implant are physical methods, particularly antimicrobial photodynamic therapy (aPDT) [1]. This method shows a pronounced photobactericidal activity paired with an anti-inflammatory effect. aPDT also prevents dystrophic and sclerotic processes helping reduce the risk of implant rejection and accelerate biointegration.

The most perspective material, which is widely used in the field of clinical implantation these days, is hydroxyapatite. Hydroxyapatite is characterized by high stability, bioactivity, and biocompatibility [2]. Photosensitizers in nano form can be used as active photobactericidal substances for implant coatings, which do not exhibit their photodynamic activity in the absence of inflammatory agents [3]. It is hypothesized that meso-tetra(3-pyridyl)bacteriochlorin nanoparticles (Bch NPs) and non-sulfonated aluminum phthalocyanine nanoparticles (AlPhc NPs) are the most promising photosensitizers, especially in the monitoring of pathological processes with a deep localization in the implant [4, 5]. Therefore (Bch NPs) and AlPhc NPs were investigated as photosensitizers that can provide the greatest light and drug dose penetration depth of aPDT treatment. Test compounds have an absorption peak in the near-infrared and red ranges, respectively. Such spectral features of the drug correspond to the maximum optical transparency region of biological tissues.

2 Materials and methods

The luminescence spectra of Bch NPs and AlPhc NPs were examined using a fiber spectrometer LESA-01-BIOSPEC [6] in the range of 400–1100 nm in various conditions, including the interaction with surface hydroxyapatite molecules. The excitation of the NPs was realized by laser radiation at a power density of ~100 mW/cm² and wavelengths of λ=532 nm and 632.8 nm which were selected in accordance with the photosensitizers’ spectral absorption maximums.

3 Results

The analysis of the luminescence spectra dynamics for both types of crystalline NPs have shown that initially not photoactive photosensitizers’ nanocrystals acquire the ability to luminescence due to an interaction with hydroxyapatite surface molecules. However, the luminescence peaks intensity varies over time under the influence of the exciting laser radiation (Figure 1).

Figure 1:

The dynamics of the luminescence spectra time transforming of the implant based on hydroxyapatite coated by (A) Bch NPs (λ_ex=532 nm) and (B) AlPhc NPs (λ_ex=632.8 nm).

4 Conclusion

The possibility of the NPs’ activation on the surface of covered implants was proved during the study. The activity level of NPs was estimated by the control of the photoluminescence intensity. Based on the research it can be concluded that the photosensitizers’ NPs interact both among themselves and with a complex porous structure of the implant. The findings of the study suggest that this technology is promising in order to create implants with photobactericidal properties.

References

[1] Haag PA, Steiger-Ronay V, Schmidlin PR. The in vitro antimicrobial efficacy of PDT against periodontopathogenic bacteria. Int J Mol Sci 2015;16(11):27327–38.

[2] Nandi SK, Kundu B, Mukherjee J, Mahato A, Datta S, Balla VK. Converted marine coral hydroxyapatite implants with growth factors: In vivo bone regeneration. Mater Sci Eng C Mater Biol Appl 2015;49:816–23.

[3] Ryabova A, Steiner R, Loschenov VB. Metamorphosis of crystalline nanoparticles consist of photosensitizers molecules in biotissues. Abstract of the International Conference on Laser Applications in Life Sciences (LALS), 29 June – 2 July 2014, Ulm, Germany. http://lals2014.ilm-ulm.de/fileadmin/file_uploads/LALSAbstractband.pdf [Accessed on September 21, 2016].

[4] Vasilchenko SY, Volkova AI, Ryabova AV, Loschenov VB, Konov VI, Mamedov AA, Kuzmin SG, Lukyanets EA. Application of aluminum phthalocyanine nanoparticles for fluorescent diagnostics in dentistry and skin autotransplantology. J Biophotonics 2010;3(5–6):336–46.

[5] Oertel M, Schastak SI, Tannapfel A, Hermann R, Sack U, Mössner J, Berr F. Novel bacteriochlorine for high tissue-penetration: photodynamic properties in human biliary tract cancer cells in vitro and in a mouse tumour model. J Photochem Photobiol B 2003;71(1-3):1–10.

[6] Loschenov VB, Konov VI, Prokhorov AM. Photodynamic therapy and fluorescence diagnostics. Laser Phys 2000;10(6):1188–207.

[1.02] Quantum medicine: Molecular appearance

Galina A. Zalesskaya and Ludmila G. Astaf’eva

B. I. Stepanov Institute of Physics, NASB, 68 Nezavisimosti Ave., Minsk, 220072, Belarus

E-mail contact: zalesskaya@imaph.bas-net.by

Abstract: The effects of phototherapy on blood oxygenation and metabolic processes were studied. It was shown that blood irradiation exerts an influence on the oxygen exchange and formation of reactive oxygen species, regulating many processes in the living organism.

1 Introduction

The purpose of this research is to study molecular mechanisms of low-intensity optical radiation (OR) action on patients’ blood irradiated in vivo by different wavelengths (254, 632.8, 670, and 780 nm).

2 Materials and methods

About 200 blood samples were analyzed after extracorporeal, intravenous or over-vein blood irradiation. Objects of investigation were the spectral characteristics of venous blood samples, the degree of hemoglobin oxygenation in venous (S_VO₂) and arterial blood, the blood gas composition, especially the partial pressure of oxygen (p_VO₂), the acid-base balance indices, the hemoglobin concentration as well as the content of some metabolic products. The dynamics of blood oxygenation during a course of phototherapy (PT) was determined. Taking into account the optical properties of human tissues, the depth penetration of OR in blood and skin tissue were evaluated in the spectral range of 405–950 nm, and the efficiency of action was compared between the OR provided by different wavelengths.

3 Results

It could be observed that quantitative differences in the oxygenation of the blood in various patients depended primarily on the original content of hemoglobin fractions and optical characteristics of blood at the wavelengths studied. The investigations showed that, the irradiation of blood by therapeutic doses of OR for the wavelengths used, initiated the similar molecular changes in the blood and its components, and that incoherent monochromatic light acts equally efficient as laser radiation.

Differences in short- and long-term changes of the blood’s oxygenation characteristics were observed (Figure 1). An increase of p_VO₂ and a decrease of carbon dioxide partial pressure in venous blood were obtained during and immediately after blood irradiation. Reversibility of the photoinduced changes in the characteristics of blood oxygenation was noted. Blood oxygenation increased during irradiation and decreased to the beginning of the next procedure up to the level which was close to the initial level or even less. It was also seen that during the PT treatment there were positive changes in the blood oxygenation. As a rule, p_VO₂ and S_VO₂ increased up to normal values under the influence of PT and then decreased after finishing the irradiation at initial or even lower values over a time interval not exceeding 15 min. Thus, in patients with low venous blood S_VO₂ values, a normalization was obtained during irradiation.

Figure 1:

Oscillation of the oxygen content in venous blood (Ct_VO₂): 1 – before (■) and during (□) the procedure of intravenous blood irradiation (λ=670 nm) and 2 – before (●) and during (○) the procedure of ultraviolet blood irradiation (λ=254 nm). Oscillation of the lactate concentration (C_Lac.): 3 – before (■) and during (□) the procedure of intravenous blood irradiation and 4 – before (▲) and during (∆) the procedure of ultraviolet blood irradiation.

Periodic oscillation of not only the p_VO₂, S_VO₂, and Ct_VO₂ values but also the lactate, glucose, Ca²⁺ concentration and viscosity of blood occurred during PT, which indicated an immediate reaction of the body during irradiation. Analysis of the metabolic products (lactate, glucose) showed that during the course of treatment, the periodic oscillation in their concentration was observed with positive changes during a treatment course.

By the end of blood irradiation course, changes of gas composition in venous blood as well as the degree of hemoglobin saturation by oxygen proved to be varying sharply between different persons, depending on both the photoinduced changes in the S_VO₂ values and the initial values of blood oxygenation. Changes in the metabolic product concentration obtained after completion of the course depended on two quantities: their initial concentration, which decreases for high initial levels and conversely increases for low initial levels, and photoinduced changes in the level of hemoglobin oxygen saturation.

Overall, hemoglobin was considered as the primary photoacceptor during blood irradiation with OR of different wavelengths. Initial mechanisms of action observed include oxyhemoglobin photodissociation following photoexcitation of hemoglobin’s electronic states; altering the oxygenation characteristics and oxygen exchange in the body; improvement in oxygen utilization by tissues and oxygen consumption in cells; and correction of the process for reactive oxygen species (ROS) production. The biochemical effects are generated by the activation of molecular oxygen to ROS which initiates a cascade of molecular reaction, resulting in the observed therapeutic effects.

4 Conclusion

It can be concluded that absorption of OR by blood irradiated by its therapeutic doses has an effect on oxygen exchange in the body, altering the oxygenation characteristics. Consequently, the balance changes between the production of ROS and their inhibition by antioxidant systems, resulting in an intensification of metabolic processes.

[1.03] Aluminum phthalocyanine nanoparticles as a contrast agent for the detection of tooth enamel microcracks

Julia O. Kuznetsova¹, Dina S. Farrakhova¹ and Maxim G. Yassin²

¹ National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

² Dental Clinic “Prodental”, The new boulevard 21, Dolgoprudny, Moscow region, 141707, Russian Federation

E-mail contact: JOKuznetsova@mail.ru

Abstract: This paper describes the possible application of aluminum phthalocyanine nanoparticles (nAlPc) for the diagnosis, prevention, and therapy of inflammatory diseases in dentistry. It could be observed that nAlPc fluoresces in the nanoparticle formed in the presence of pathologic microflora or the inflammation process. This allows identification of local pathological microflora accumulation within microcracks of the tooth’s enamel, and diagnostics and treatment of inflammatory diseases to be performed. Experimental studies of the interaction of nAlPc with tooth enamel in the presence of different components of toothpaste were made. The nAlPc fluorescence was measured using the LESA-01-BIOSPEC fiber-optic spectrometer (BioSpec, Moscow, Russia).

1 Introduction

Application of metal phthalocyanine nanoparticles in clinical practice opens a new perspective to increase the efficacy of fluorescence diagnostics (FD) and photodynamic therapy (PDT).

Early diagnosis of tooth-enamel microcracks has a great importance in modern dentistry for caries prevention. Diagnostic methods being used in clinical dentistry, such as visual inspection, probing, and vital enamel staining [1], can only detect large-area enamel damages. However, for diagnostics it is possible to use the autofluorescence of porphyrins provided that microflora is abundant in the microcracks [2]. Otherwise, the microflora’s autofluorescence is relatively weak. The problem can be solved by using nAlPc as an exogenous fluorophore. Aluminum phthalocyanine nanoparticles are a convenient tool for FD because they do not fluoresce in the nanoparticle form but in the monomeric form. The fluorescence occurrs when AlPc molecules are arranged vertically attached to the surface of the nanoparticles during the dissolution process or the interaction with pathological tissue, pathologic microflora and macrophages [3, 4].

However it is not only possible to use nAlPc for FD but also for antimicrobial PDT [5]. Aluminum phthalocyanine nanoparticles are a promising photosensitizer for clinical use because they have both a good transportation in aqueous media and penetration into the tissue.

For feasable use in dentistry, a colloidal nAlPc solution could be employed for detection of local accumulation of pathological microflora on the enamel surface and in the microcracks area. There is also the possibility of applying nAlPc by adding them to different components of toothpaste, which is discussed in the present paper.

2 Materials and methods

Human teeth, extracted for different reasons, including periodontal problems, were used for investigation of application options of nAlPc for enamel microcracks diagnosis. After extraction, all samples were placed into tubes with a 0.9% sodium chloride solution. Before the experiment teeth were mechanically cleaned and rinsed in running water.

For the diagnosis of enamel microcracks by detecting nAlPc fluorescence in the areas of accumulation of pathogenic microflora it was planned to design special toothpaste with nAlPc. For the creation of this composition with nanoparticles, all paste components were examined separately. Experimental samples containing 100 mg of the test component were prepared to hold 500 μl of the colloidal nAlPc solution in a concentration of 50 mg/l. Fluorescence spectra measurements were performed 96 h after their preparation to account for the possible increase in the interaction between the component and nAlPc. Then the base toothpaste composition was created. From the spectroscopic point of view, this composition was an almost entirely ‘black body’.

For the fluorescence measurements, a fiber-optic spectrometer LESA-01-BIOSPEC (BioSpec, Moscow, Russia) was used. A helium-neon laser with a wavelength of 632.8 nm and an output power of 5 mW at the fiber end was used as an irradiation source for fluorescence excitation. A fiber-optic probe with one illuminating and six receiving fibers (each of 200 µm in diameter) was attached to the spectrometer. Detailed information about the experimental set-up can be found in [6].

3 Results

Autofluorescence investigation of the hard tissues of the teeth showed that considerable demineralization of enamel led to a greater intensity of the fluorescence of the enamel. Conversely, the higher the degree of mineralization of enamel, the lower the enamel autofluorescence intensity. During experiments, it could be observed that autofluorescence wavelengths of carious enamel are shifted to the red region of the spectrum. Fluorescence spectra of nAlPc were obtained from regions on the enamel surface with microcracks after applying nanoparticles on the entire enamel surface. It could also be seen that over time there is a nAlPc fluorescence amplification in the areas of enamel microcracks. This indicates the presence of bacteria in these areas.

For the prevention and early diagnosis of caries, a basic toothpaste composition containing nAlPc at concentrations of 10, 100 and 1000 mg/l was investigated. Research revealed that for the base toothpaste composition, containing particles in concentrations of 10 and 100 mg/l, the fluorescence was absent or weak. The base toothpaste composition in a concentration of 1000 mg/l was not only applied to the enamel surface microcracks but also to the affected areas of the enamel caries. Strong nAlPc fluorescence occurred only in the areas of carious lesions and was weak in the areas with a low content of bacteria.

4 Conclusion

The nAlPc included in toothpaste composition could be used as an exogenous fluorophore for detection of a local accumulation of pathological microflora on the enamel surface and in microcracks in dentistry. Future additional research is necessary pertaining to the selection of the optimal toothpaste composition and nAlPc concentration to more accurately diagnose localization of microcracks on the tooth enamel surface.

Acknowledgments: This work was supported by the Competitiveness Programm of NRNU MEPhI. The research was carried out in the Laser Biospectroscopy Laboratory at the A. M. Prokhorov General Physics Institute of RAS together with the Laser Micro and Nanotechnology Department of NRNU MEPhI. We thank Prof. Victor B. Loschenov for his scientific support.

References

[1] Cobb CM. Lasers in periodontics: a review of the literature. J Periodontol 2006;77(4):545–64.

[2] Kuznetsova JO, Scheib G, Trottmann M, Leeb R, Sroka R, Homann C. OCT imaging-characterization and medical applications. 16th International Conference “Laser Optics 2014”, June 30 – July 4, 2014, St. Petersburg, Russia. http://www.gpi.ru/3rd_Inter_Symp_LM_14.html [Accessed on September 15, 2016].

[3] Breymayer J, Rück A, Ryabova AV, Loschenov VB, Steiner RW. Fluorescence investigation of the detachment of aluminum phthalocyanine molecules from aluminum phthalocyanine nanoparticles in monocytes/macrophages and skin cells and their localization in monocytes/macrophages. Photodiagnosis Photodyn Ther 2014;11(3):380–90.

[4] Vasilchenko SYu, Volkova AI, Korovin SB, Loschenov VB, Sinyaeva ML, Mamedov AA, Lukyanets EA., Kuzmin SG. Investigation of aluminium phthalocyanine nanoparticles fluorescence properties in tooth enamel microdamages (only in Russian). Photodyn Ther 2006;5(2):77–80. http://cyberleninka.ru/article/n/issledovanie-flyuorestsentnyh-svoystv-nanochastits-ftalotsianina-alyuminiya-v-mikropovrezhdeniyah-emali-zuba [Accessed on September 15, 2016].

[5] Asem H, El-Fattah AA, Nafee N, Zhao Y, Khalil L, Muhammed M, Hassan M, Kandil S. Development and biodistribution of a theranostic aluminum phthalocyanine nanophotosensitizer. Photodiagnosis Photodyn Ther 2016;13:48–57.

[6] Loschenov VB, Konov VI, Prokhorov AM. Photodynamic therapy and fluorescence diagnostics. Laser Phys 2000;10(6):1188–207.

[1.04] Singlet oxygen generation by zeolite-porphyrin complexes

Marina V. Parkhats¹, Sergei V. Lepeshkevich¹, Alexander S. Stasheuski¹, Boris M. Dzhagarov¹, Hakob H. Sargsyan², Robert K. Ghazaryan³, Anna G. Gyulkhandanyan² and Grigor V. Gyulkhandanyan²

¹ B. I. Stepanov Institute of Physics, NASB, 68 Nezavisimosti Ave., Minsk, 220072, Belarus

² Institute of Biochemistry, National Academy of Sciences of Armenia, 5/1 P. Sevak str., Yerevan, 0014, Armenia

³ Yerevan State Medical University, 2 Koryun str., Yerevan, 0025, Armenia

E-mail contact: m.parkhots@ifanbel.bas-net.by

Abstract: The investigated zeolite-porphyrin complexes generate singlet oxygen with low quantum yields and can not be used as photosensitizers (PS) for photodynamic therapy (PDT).

1 Introduction

Many strategies have been developed to improve efficiency of PDT, a form of phototherapy involving light and PS to treat tumors. Photosensitizer incorporation into different types of nanoparticles is one of such strategies allowing to improve tumor targeting and to minimize the side effects of PS. However, PS incorporation can reduce photodynamic activity of PS by changing its photophysical properties and singlet oxygen generation.

In the present work, nanozeolites were used as carriers for PS. Zeolites are crystalline aluminosilicates consisting of enclosed regular cavities and channels of well-defined size and shape, that are widely used in ion exchange technology, catalysis, filtering and gas adsorption. It is also known that natural zeolite clinoptilolite possesses antitumor activity based on a stimulation of the immune system [1].

The aim of the work was to investigate photosensitized singlet oxygen generation by nanozeolite-porphyrin complexes and their photophysical properties.

2 Materials and methods

Nanoparticles made of natural zeolite clinoptilolite, from the Noyemberyan region in the Republic of Armenia, with nanometric sizes of 80–150 nm, and cationic porphyrins such as Zn-meso-tetra(4-N-hydroxyethyl-pyridyl) porphyrin (ZnTOEt4PyP) and meso-tetra(4-N-hydroxyethyl-pyridyl) porphyrin (TOEt4PyP) (Figure 1) were used as carriers and PS, respectively. Zeolite-porphyrin complexes were obtained by adding of small amounts of porphyrin to a nanozeolite solution. Centrifugation was used to remove unbound porphyrin molecules. Absorption spectra were recorded using a spectrophotometer Cary-500 Scan. Kinetics of the triplet-triplet absorption were measured using a nanosecond laser spectrometer [excitation wavelength, 532 nm; Nd:YAG laser (LS-2132U, LOTIS TII, Belarus); pulse width, 8 ns; repetition rate, 13 Hz]. Time-resolved singlet oxygen luminescence in the near-infrared (NIR) region was recorded with a nanosecond laser NIR spectrometer [excitation wavelength, 532 nm; Nd:YAG laser (DTL-314QT, Laser-export Co. Ltd., Moscow, Russia); pulse width, 10 ns; pulse energy, 1 μJ; repetition rate, 2.5 kHz). To determine quantum yields of photosensitized singlet oxygen generation (γ_Δ), TOEt4PyP in an aqueous solution (γ_Δ=0.77) was used as a standard [2]. All experiments were carried out in distilled water at room temperature.

Figure 1:

Structural formulas of ZnTOEt4PyP (M=Zn) and TOEt4PyP (M=2H).

3 Results

It could be shown that complexation of the porphyrins ZnTOEt4PyP and TOEt4PyP leads to transformations and red shifts of the absorption and fluorescence spectra. Significant broadening and changes in the relative intensity of the Soret band were observed. The interaction of both porphyrins with nanozeolite causes also fluorescence quenching. Kinetic absorption studies showed that the kinetics of triplet-triplet absorption of nanozeolite-porphyrin complexes has a two-exponential character. The obtained photophysical properties of nanozeolite-porphyrin complexes indicate the presence of an inhomogenity in porphyrin interaction with nanozeolite. Moreover, conformational changes and aggregation effects upon adsorption of cationic porphyrins on anionic [AlSi]O₄⁻ framework of nanozeolite can not be excluded.

The kinetics of singlet oxygen luminescence photosensitized by porphyrins, nanozeolite-porphyrin complexes as well as the signal of nanozeolite in an aqueous solution were investigated. The results obtained show that the kinetic of singlet oxygen luminescence photosensitized by nanozeolite-porphyrin complexes not have the rise component that is always observed for PS in solutions (Figure 2). The kinetics of singlet oxygen luminescence photosensitized by nanozeolite-porphyrin complexes are well fitted by a biexponential decay function with τ₁~300 ns (fast component) for both nanozeolite-porphyrin complexes, and τ₂=6.6±0.5 µs and 9.8±0.8 µs (slow component) for nanozeolite-TOEt4PyP and nanozeolite-ZnTOEt4PyP, respectively (Figure 2). Based on the control measurements of nanozeolite without porphyrins, it could be concluded that the fast component τ₁ characterizes luminescence of nanozeolite. On the other hand, based on the spectral properties of the detected signal, the slow component τ₂ can be unambiguously attributed to the luminescence of singlet oxygen.

Figure 2:

Kinetics of singlet oxygen luminescence photosensitized by nanozeolite-TOEt4PyP complex in aqueous solution. Concentrations of TOEt4PyP and nanozeolite were 10 μM and 0.6 mg/ml, respectively. Excitation wavelength, λ_exc=532 nm; detection wavelength, λ_det=1270 nm.

Quantum yields of photosensitized singlet oxygen generation by nanozeolite-porphyrin complexes were found to not exceed 0.04 and 0.01 for nanozeolite-TOEt4PyP and nanozeolite-ZnTOEt4PyP, respectively. Such low quantum yields of singlet oxygen generation can be explained by (i) the porphyrin aggregation upon interaction with nanozeolite, and (ii) by the quenching of singlet oxygen by alumina anions from [AlSi]O₄⁻ framework of nanozeolites [3]. It should be noted that the refractive index of zeolite (n=1.47–1.48) is larger than that of water (n=1.33). Taking into account the dependence of radiative rate constant of singlet oxygen deactivation (k_r) on the refractive index (n) [4], it can be expected that k_r in zeolite is 3 times greater than the one in water. Therefore, the above mentioned values of the quantum yields of singlet oxygen generation in nanozeolite can be considered as the upper limits of γ_Δ.

4 Conclusion

It was demonstrated that the complexes of nanozeolite-TOEt4PyP and nanozeolite-ZnTOEt4PyP generate singlet oxygen with very low quantum yields and therefore can not be used as PS for PDT.

Acknowledment: This work was supported by the Foundation of Basic Research of the Republic of Belarus (Grant numbers: ‘Ph14Arm-015’ and ‘Ph15SO-036’).

References

[1] Pavelić K, Hadzija M, Bedrica L, Pavelić J, Dikić I, Katić M, Kralj M, Bosnar MH, Kapitanović S, Poljak-Blazi M, Krizanac S, Stojković R, Jurin M, Subotić B, Colić M. Natural zeolite clinoptilolite: new adjuvant in anticancer therapy. J Mol Med (Berl) 2001;78(12):708–20.

[2] Stasheuski AS, Galievsky VA, Knyukshto VN, Ghazaryan RK, Gyulkhandanyan AG, Gyulkhandanyan GV, Dzhagarov BM. Water-soluble pyridyl porphyrins with amphiphilic n-substituents: fluorescent properties and photosensitized formation of singlet oxygen. J Appl Spectrosc 2014;80(6):813–23.

[3] Jockusch S, Sivaguru J, Turro NJ, Ramamurthy V. Direct measurement of the singlet oxygen lifetime in zeolites by near-IR phosphorescence. Photochem Photobiol Sci 2005;4(5):403–5.

[4] Dzhagarov BM, Jarnikova ES, Parkhats MV, Stasheuski AS. Dependence of the spontaneous emission of singlet oxygen on the refractive index and molecular polarizability of the surrounding dielectric media. Opt Spectrosc 2014;116(6):926–32.

[1.05] Detection of flavin fluorescence in lung adenocarcinoma cells by FLIM

Ekaterina A. Boruleva^{1, 2}, Victoria V. Zherdeva² and Alexander P. Savitsky²

¹ National Research Nuclear University (NRNU), Moscow Engineering Physics Institute (MEPhI), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

² A. N. Bach Institute of Biochemistry of RAS, Leninsky prospekt 33, build. 2, Moscow, 119071, Russian Federation

E-mail contact: ipcoova@yandex.ru

Abstract: Changes in endogenous fluorescence can serve as an indicator of changes in biochemical status of the cells, which to date has been demonstrated for a number of endogenous fluorophores. Conditions of endogenous fluorescence detection in tumor cells were evaluated using the fluorescence-lifetime imaging microscopy (FLIM) method for determining the flavin fluorescence.

1 Introduction

The aim of our work was to visualize the endogenous fluorescence in tumor cells by FLIM and determine the sources of flavin fluorescence.

2 Materials and methods

FLIM was performed using a time-resolved confocal fluorescence microscope (MicroTime 200, PicoQuant GmbH, Berlin, Germany). A solid-state 473-nm laser was used for excitation (PicoQuant GmbH), a 500/14–25 nm filter (Semrock Inc., Rochester, USA) for fluorescence detection. The fluorescence spectra were detected using an Andor chamber (PicoQuant GmbH). The images were processed using the PicoHarp and SymPhoTime software (PicoQuant GmbH).

3 Results

The lifetime distribution of autofluorophors in tumor cells was obtained. It was shown that the signal was distributed unevenly, originating from the organelle-morphed structure. Parts of these organelles were colored by the specific mitochondria dye MitoTracker^® Orange (Thermo Fisher Scientific, Waltham, USA). Fluorescence decay curves were approximated well by a double exponential model with an average lifetime of 2.3 ns. This presumably corresponded to the lifetime of flavins.

4 Conclusion

The results can be applied for studies of cell biochemistry in real-time mode when the change in the level of endogenous fluorophores may be an indicator of some pathological processes.

[1.06] Spectroscopic evaluation method of angiogenesis in the healing of skin grafts using spectrally sensitive-to-inflammatory reactions aluminum phthalocyanine nanoparticles

Vladimir I. Makarov¹, Daria V. Pominova¹, Maria N. Kholodtsova^{1, 2, 3}, Anastasia V. Ryabova^{1, 4} and Victor B. Loschenov^{1, 4}

¹A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

²Université de Lorraine, CRAN, 2 Avenue de la Forêt de Haye, 54516 Vandoeuvre-Lès-Nancy cedex, France

³CNRS, CRAN, 54516 Vandoeuvre-Lès-Nancy cedex, France

⁴National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Kashirskoe Shosse 31, Moscow, 115409, Russian Federation

E-mail contact: vi.makarov@physics.msu.ru

Abstract: The development of a rapid method to assess the status of a skin graft, i.e., the tissue components involved in the healing of the affected skin, or rejection of skin grafts, was carried out by spectroscopic means. In order to assess the extent of inflammation, aluminum phthalocyanine nanoparticles (NP-AlPc) were employed.

1 Introduction

During transplantation of any tissue, acute rejection is a major cause of functional failure of the graft, and it is a major risk factor for both graft lifetime and life per se. Currently, the only accurate method of predicting possible transplant rejection (which allows determiniation of rejection, as well as its type) is a biopsy. A search is being carried out for new photosensitizers based on nanotechnology for this purpose.

The proposed method for assessing the state of skin spectroscopically is to assess their component properties using locally applied NP-AlPc. The NP-AlPc provides information about the physiological condition of the skin and allows assessment of the degree and rate of engraftment or rejection. Additionally, it enables control of several biochemical and physiological parameters in the entire graft, or the whole area of the skin lesions. Such parameters include the oxygenation of hemoglobin in the tissue microvasculature; the blood supply level; blood flow and lymph flow; assessment of intracellular metabolism; and assessment of the cellular respiration (aerobic vs. anaerobic). The method offers a prime opportunity to determine all of these settings for monitoring in real time.

2 Materials and methods

The experiment was performed in vivo in five outbred mice. Cross-skin transplantation was carried out on the back of each mouse. A colloidal solution of NP-AlPc was added under the autograft located on the right, whilst the other half remained unmodified to enable autograft engraftment monitoring without the nanoparticles.

The level of hemoglobin oxygenation and blood filling of the tissue grafts was evaluated using diffuse backscattering spectroscopy [1], with a tungsten halogen lamp as a light source. Assessment of the cellular respiration type of the transplant during engraftment (aerobic vs. anaerobic) was carried out using fluorescence spectroscopy [2] by measuring the luminescence spectra of the oxidized and reduced nicotinamide adenine dinucleotide (NAD). The third harmonic of a pulsed Nd: YAG laser was used as the radiation source (λ=355 nm, τ_pulse=7 ns, ν=15 Hz).

The condition of blood and lymph flow in the autografts was assessed by analyzing the recorded fluorescence images using the exogenous photosensitizers and fluorescent dyes (Photosens and indocyanine green) that absorb in the red and near-infrared spectral range for deeper excitation light penetration into the skin. Registration of fluorescence images was performed using a video system that consists of a laser source, a broadband source diode, a beam splitter with a dichroic mirror and two digital cameras for recording color images and luminescence.

To evaluate the intensity of the immune response in the transplant engraftment, a time-resolved fluorescence spectroscopy method was used in the pico- and nanosecond range in combination with the NP-AlPc, spectrally sensitive to inflammatory reactions [3, 4].

3 Results and discussion

Figure 1 shows the results of hemoglobin oxygenation measurements in the skin grafts from two mice on day 7 after transplantation. All the transplants on the right, containing nanoparticles, have the same degree of oxygenation i.e. three times lower than the norm, which indicates the beginning of the healing process. The grafts on the left differ from each other and, as can be seen from the figure, one of the grafts has critically low degree of oxygenation, which may indicate an unfavorable prognosis with respect to engraftment. Figure 2 shows the fluorescence spectra of reduced NAD. The calculated concentration of NAD in the transplant without the nanoparticles is 2.5 times higher than transplants with NP-AlPc or the healthy tissue. This is most probably due to the anaerobic cell respiration caused by limited access to the circulatory system, which can also indicate an unfavorable engraftment prognosis.

Figure 1:

Oxygenation of hemoglobin in different parts of the skin.

Figure 2:

NADH fluorescence spectra.

Figure 3A shows the spread of indocyanine green a few minutes after its administration in mouse skin tissue, 7 days after cross skin transplantation. It can be seen that the grafts fluoresce less than the healthy skin. This means that at this stage, new blood vessels have not yet developed in the graft tissue. Evaluation of lymph flow was made on the analysis of fluorescence images of skin grafts containing activated NP-AlPc (Figure 3B). A NP-AlPc interaction study with macrophages showed that NP-AlPc fluorescence intensity increases in the inflammation area.

Figure 3:

Fluorescent images of skin grafts with indocyanine green (A) and NP-AlPc (B).

Figure 4 shows the dependence of the right graft fluorescence intensity on the photodynamic treatment duration at λ=670±5 nm and a power density of 0.5 W/cm². It can be seen that there is distinct photobleaching, which is not observed when using molecular forms of AlPc. It was also found that NP-AlPc interaction with monocytes and macrophages leads to changes in their fluorescence lifetime.

Figure 4:

Fluorescence intensity at different photodynamic treatment durations.

Acknowledgment: This work was supported by Russian Foundation for Basic Research (Grant number: ‘15-29-04869-ofi-m’).

References

[1] Stratonnikov AA, Loschenov VB. Evaluation of blood oxygen saturation in vivo from diffuse reflectance spectra. J Biomed Opt 2001;6(4):457–67.

[2] Sanchez WY, Pastore M, Haridass IN, König K, Becker W, Roberts MS. Fluorescence lifetime imaging of the skin. In: Becker W, editor: Advanced time-correlated single photon counting applications. Springer series in chemical physics 111. Cham, Heidelberg, New York, Dordrecht and London: Springer; 2015, p. 457–508.

[3] Bystrov FG, Makarov VI, Pominova DV, Ryabova AV, Loschenov VB. Analysis of photoluminescence decay kinetics of aluminum phthalocyanine nanoparticles interacting with immune cells. Biomed Photon 2016;5(1):3–8.

[4] Makarov VI, Vasil’chenko SYu, Ryabova AV, Loschenov VB. Use of optical-spectral methods for in vivo non-invasive assessment of nanoparticles accumulation in biological tissues. Russ J Gen Chem 2015;85(1):341–5.

[1.07] Time-resolved laser-induced fluorescence spectroscopy for identification of pituitary adenoma

Andrey N. Sobchuk¹, Nicolai A. Nemkovich¹, Yulia V. Kruchenok¹, Yury G. Shanko² and Andrey I. Chuhonsky²

¹B. I. Stepanov Institute of Physics, NASB, 68 Nezavisimosti Ave., Minsk, 220072, Belarus

²Republican Research and Clinical Center of Neurology and Neurosurgery, Ministry of Health of the Republic of Belarus, 24 F. Skoriny Str., Minsk, 220114, Belarus

E-mail contact: sobchuk@ifanbel.bas-net.by

Abstract: Rapid and high-sensitivity identification of pituitary adenoma can be carried out by measuring the autofluorescence decays. A significant difference in the autofluorescence mean lifetimes of tumorous and healthy tissues in the 380–600-nm spectral range was observed.

1 Introduction

It is well-known that human tissues contain biomolecules that fluoresce well in the ultraviolet (UV), visible, and near-infrared regions because they contain endogenous chromophores. The latter include tryptophan, tyrosine, dinucleotides, collagen, flavins, lipofuscins, porphyrins, etc. The characteristics of the intrinsic fluorescence of the chromophores depend on their distribution in the tissues, the concentration of ions, the properties of the microenvironment, and other factors. The appearance of a pathological process affects the physicochemical microcharacteristics of the tissues and therefore changes the autofluorescence parameters of the tumors.

Steady-state and time-resolved measurement methods are used for the fluorescence analysis of tissues. Steady-state measurements, as a rule, involve recording of the luminescence intensity at a definite wavelength, the emission and excitation spectra, and the emission anisotropy. The main drawbacks of steady-state measurements are that the fluorescence intensity depends on the optical excitation and emission layout, the surface inhomogeneity of the tissues, different concentrations of the chromophores, and the presence of endogenous absorbers in the tissues, especially hemoglobin and oxyhemoglobin. Time-resolved diagnostic methods are largely free of the drawbacks inherent to steady-state spectroscopy, because endogenous absorbers only attenuate the intensity of the intrinsic fluorescence of the tissues; i.e., they act as an internal filter but do not change the character and duration of the luminescence decay, as well as weakly affect the kinetics of the autofluorescence spectra. It should be pointed out that it is convenient to analyze and to statistically process the values of the preexponential factors and fluorescence lifetimes of the separate components of the luminescence decay when it is resolved into exponentials.

2 Materials and methods

Measurements were made on samples of healthy and tumorous tissues of the pituitary taken after an operation carried out at the Republican Research and Clinical Center of Neurology and Neurosurgery, Ministry of Health of the Republic of Belarus. The tissue samples were kept into 0.9% physiological solution and were investigated a few hours after being harvested. The presence of a tumor was estimated macroscopically immediately after taking the sample, and microscopically based on the results of a histological examination. Nineteen tissue samples were investigated, from which there were eight samples of pituitary adenoma and eleven samples of healthy pituitary tissue.

The system for exciting and detecting the autofluorescence decays consists of a HORIBA PicoBrite pulsed semiconductor light emitting diode (LED) (emission wavelength, 342 nm; pulse width at half height, 700 ps; pulse repetition rate, 10 MHz), a SOLAR ML-44 monochromator (inverse linear dispersion, 18.7 nm/mm), a Hamamatsu H5773 photomultiplier (recording range, 185–820 nm; time resolution, 180 ps) and a Becker&Hickl SPC-130 time-correlated photon-counting module. The mean emission power of the semiconductor LED during the experiments at a pulse repetition rate of 10 MHz was 1 μW, whereas the time to record the autofluorescence decay was 3 min. The fluorescence was excited and recorded via an Avantes optical-fiber probe FCR-7UV400-2-ME, which consists of one central optical fiber, 400 μm in diameter, for transporting the excitation light from the LED to the tissue and six optical fibers of the same diameter located around the central optical fiber for recording the emission signal. During the measurements the probe was 1–2 mm away from the tissue surface, and the light spot was 2 mm in diameter.

3 Results

Our studies showed that the autofluorescence kinetics of the tissues in the spectral range of 380–600 nm does not have an exponential character. From representing the kinetics as a superposition of exponentials it follows that the contribution to the autofluorescence includes two sub-nanosecond components with lifetimes of 0.39–0.53 and 1.9–2.5 ns respectively, and a slower nanosecond component with a fluorescence lifetime of 6.9–8.2 ns. The mean lifetimes of the short-lived components are less in tumorous tissue than in healthy tissue, and the mean lifetimes of the slower nanosecond component is about the same in the various objects of investigation.

Figure 1 illustrates how the autofluorescence mean lifetime of the tissues depends on the emission wavelength. It can be seen that the autofluorescence mean lifetime of healthy and tumorous tissues initially increases with increasing wavelength to about 500 nm and then begins to decrease. The difference between the autofluorescence mean lifetime of tumorous and healthy tissues monotonically increases with increasing wavelength and, when recorded at 600 nm, reaches the highest value of 1.6 ns.

Figure 1:

Mean autofluorescence lifetime τ of healthy pituitary tissue () and tissue of a pituitary adenoma (■).

Discriminant analysis was used to analyze the data obtained here. Autofluorescence mean lifetime at emission wavelengths of 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580 and 600 nm were selected as discriminant variables. The calculated values of the discriminant functions are shown in Figure 2. The sensitivity and specificity of the identification of pituitary adenoma, determined by means of discriminant analysis, is 100%.

Figure 2:

Values of the discriminant function of healthy () and tumorous (■) tissues.

4 Conclusion

Rapid and high-sensitivity identification of pituitary adenoma can be carried out by measuring the autofluorescence decays at emission wavelengths of 380–600 nm. The method could be further improved by using excitation wavelengths of about 260 nm, which would make it possible to additionally record the UV autofluorescence of tyrosine and tryptophan.

[1.08] High-efficiency stimulated low-frequency Raman scattering in water/buffer suspension of potato viruses (PVX & PVA)

Aleksey F. Bunkin¹, Mikhail Ya. Grishin², Olga V. Karpova³, Anna D. Kudryavtseva⁴, Vasily N. Lednev⁵, Tatyana V. Mironova⁴, Sergey M. Pershin¹, Ekaterina K. Petrova³, Maksim A. Strokov⁴, Nikolay V. Tcherniega⁴ and Konstantin I. Zemskov⁴

¹A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

²Moscow Institute of Physics and Technology (State University), 9 Institutskiy per., Dolgoprudny, Moscow Region, 141701, Russian Federation

³Lomonosov Moscow State University, Vorob’evy Gory 1, Moscow, 119991, Russian Federation

⁴Lebedev Physical Institute of RAS, Leninsky pr. 53, Moscow, 119991, Russian Federation

⁵National University of Science and Technology MISiS, Leninsky Ave 4, Moscow, 119991, Russian Federation

E-mail contact: akudr@sci.lebedev.ru

Abstract: Stimulated low-frequency Raman scattering (SLFRS), caused by the interaction of ruby laser pulses with the vibration modes of potato viruses X (PVX) in Tris-HCl pH 7.5 buffer and A (PVA) in water suspension was investigated. The frequency shift (on the GHz scale), efficiency conversion (up to 10%) and SLFRS threshold were measured.

1 Introduction

Viruses of any type are good examples of highly monodispersive systems. Most of the viruses have cylindrical or spheroidal shape and have their own discrete set of acoustic eigenfrequencies. Low-frequency Raman scattering (LFRS) [1, 2] can be used for their investigation. LFRS is an inelastic light scattering by acoustic vibration modes of nano or sub-micron particles. Currently, LFRS is applied in systems containing metal, dielectric, or semiconductor nanoparticles [3]. Systems consisting of viruses of different types are also the subject of intensive theoretical and experimental research [4–6]. The low-frequency spectrum of the inelastically scattered light in rigid biological structures, as in any nanoparticles system, can not only give critical information about their shape and elastic properties but can also be used for size distribution in the system under consideration. Knowledge of the exact eigenfrequency value is critical for virus identification. This important application can be realized for external incident frequencies matching the virus’ eigenfrequency. As external influence, ultrasound or electromagnetic radiation of the proper frequency can be used. Another way for effective realization is by biharmonic pumping – electromagnetic radiation containing two spectral components separated by the frequency corresponding to the virus’s eigenfrequency. For sub-micron and nanoscale structures typical eigenfrequencies are in the GHz and THz range, respectively. For biharmonic pumping, stimulated low-frequency Raman scattering (SLFRS) can be used. SLFRS is an analog of spontaneous LFRS [7, 8]. SLFRS can be excited in nano or sub-micron particles system under pulsed laser irradiation when its threshold is exceeded. SLFRS was observed for rod-like tobacco mosaic virus (TMV) [9]. The present contribution reports on the first experimental realization of SLFRS in potato viruses PVX and PVA in aqueous and Tris-HCl pH 7.5 buffer suspensions. In order to take the elastic virus environment properties into consideration, the frequency shift of stimulated Brillouin scattering (SBS) in Tris-HCl pH 7.5 buffer and water was also measured.

2 Materials and methods

Potato viruses PVX and PVA in water and Tris-HCl pH 7.5 buffer suspensions were used as samples.

Potato virus X is a type member of the Potexvirus genus (Alphaflexiviridae family). The genomic 5′-capped positive-strand ribonucleic acid (RNA), 6435-nt long, is encapsulated in a flexuous filamentous particle, about 515 nm long and 13.5 nm in diameter. Virion Mr is about 3.5×10⁶ Da, S_20w is 115–130S, buoyant density in CsCl is 1.31 g/cm³. A single linear positive-sense RNA represents about 6% by weight of the virion. About 1300 identical coat protein (CP) sub-units in PVX particle form a helical array (3.6-nm pitch) with the viral RNA packed between the turns of the helix. There are 8.9 CP sub-units (each consisting of 236 amino acid residues) per turn of the primary helix and five nucleotides are associated with each protein sub-unit. The RNA backbone is at a radial position of 3.3–3.5 nm [10].

Potato virus A is a single-stranded positive-sense RNA virus and a member of the family Potyviridae, the largest and one of the most economically important groups of plant viruses. The virions of potyviruses are flexuous and rod-shaped, 680–900 nm long and 11–15 nm wide, made up of about 2000 units of a single structural protein (coat protein, CP) encapsidating a single molecule of positive single-stranded (ss) RNA of approximately 10 kb. The particles of PVA are approximately 730 nm long and 15 nm wide. The genome is 9565 nucleotides long and contains a single open reading frame encoding a polyprotein of 3059 amino acids (aa). The PVA coat protein comprises 269 amino acids and has a calculated Mr of 30,257. The potyviral RNA strand has a poly-A tail at the 3´end and a viral genome-linked protein VPg at the 5´end [11].

Tris or tris(hydroxymethyl)aminomethane is an organic compound with the formula (HOCH₂)³CNH₂. In biochemistry and molecular biology, Tris is widely used as a component of buffer solutions including as a storage buffer for viral preparations. Tris has a pKa of 8.07 at 25°C, which implies that the buffer has an effective pH range between 7.5 and 9.0. The useful buffer range for Tris (7–9) coincides with the physiological pH typical of most living organisms. The pKa declines approximately 0.03 units per °C rise in temperature.

Ruby laser pulses (λ=694.3 nm, τ=20 ns, E_max=0.3 J) were used as an excitation source. Excitation light was focused by a lens with 50 mm focal length into the quartz cuvette filled with a viral suspension. The length of the quartz cuvette was 1 cm. The energy of the laser pulse radiation passing through and reflected from the cuvette was measured simultaneously by calibrated photodiodes. Fabri-Perot interferometers were used for spectral structure investigations, whereby the range of dispersion was changed from 0.42 to 16.7 cm⁻¹. All measurements were realized both for forward and backward directions (for the light passing through the sample and for the light reflected from it). SLFRS was experimentally measured for PVA in water suspension and for PVX in Tris-HCl pH 7.5 buffer.

3 Results

For PVA in water suspension, two Stokes components in the forward direction and one Stokes component in backward direction were recorded. A SLFRS frequency shift of 0.28 cm⁻¹ was found for the first Stokes component and 0.6 cm⁻¹ for the second Stokes component. At the experimental conditions, the threshold value of SLFRS excitation was 0.03 GW/cm².

For PVX in Tris-HCl pH 7.5 buffer, also two Stokes components in the forward direction and one Stokes component in backward direction were recorded. SLFRS frequency shift was found to be 0.21 cm⁻¹ for the first Stokes component and 0.4 cm⁻¹ for the second Stokes component. The threshold value of SLFRS excitation was 0.035 GW/cm². The threshold for backward and forward scattering was approximately the same. Line width and divergence of SLFRS were nearly the same as the corresponding values of the laser light. Maximum conversion efficiency for both cases was about 10%. Using the experimental conditions of excitation, SBS in PVX in Tris-HCl pH 7.5 buffer and PVA in water suspension could not be registered, but were excited in Tris-HCl pH 7.5 buffer and water only, and its frequency shifts were measured.

4 Conclusion

SLFRS was excited with high-efficiency conversion in PVX in Tris-HCl pH 7.5 buffer and PVA in water suspension, and was measured both for forward and backward directions. High-efficiency conversion gives the possibility of using SLFRS as the source of the biharmonic pump for pulsed impact on virus systems with the same eigenfrequencies. SLFRS also can be used for identification of different viruses.

References

[1] Duval E, Boukenter A, Champagnon B. Vibration eigenmodes and size of microcrystallites in glass: Observation by very-low-frequency Raman scattering. Phys Rev Lett 1986;56(19):2052–5.

[2] Ivanda M, Babocsi K, Dem C, Schmitt M, Montagna M, Kiefer W. Low-wavenumber Raman scattering from CdSxSe1-x quantum dots embedded in a glass matrix. Phys Rev B 2003;67:235329.

[3] Montagna M. Brillouin and Raman scattering from the acoustic vibrations of spherical particles with a size comparable to the wavelength of the light. Phys Rev B 2008;77:045418.

[4] Talati M, Jha PK. Acoustic phonon quantization and low-frequency Raman spectra of spherical viruses. Phys Rev E Stat Nonlin Soft Matter Phys 2006;73(1 Pt 1):011901.

[5] Dykeman EC, Sankey OF, Tsen KT. Raman intensity and spectra predictions for cylindrical viruses. Phys Rev E Stat Nonlin Soft Matter Phys. 2007;76(1 Pt 1):011906.

[6] Balandin A and Fonoberov V. Vibrational modes of nano-template viruses. J Biomed Nanotechnol 2005;1(1):90–5.

[7] Tcherniega NV, Samoylovich MI, Kudryavtseva AD, Belyanin AF, Pashchenko PV, Dzbanovski NN. Stimulated scattering caused by the interaction of light with morphology-dependent acoustic resonance. Opt Lett 2010;35(3):300–2.

[8] Tcherniega NV, Zemskov KI, Savranskii VV, Kudryavtseva AD, Olenin AY, Lisichkin GV. Experimental observation of stimulated low-frequency Raman scattering in water suspensions of silver and gold nanoparticles. Opt Lett 2013;38(6):824–6.

[9] Karpova OV, Kudryavtseva AD, Lednev VN, Mironova TV, Oshurko VB, Pershin SM, Petrova EK, Tcherniega NV, Zemskov KI. Stimulated low-frequency Raman scattering in a suspension of tobacco mosaic virus. Laser Phys Lett 2016; 13(8):085701.

[10] Atabekov J, Dobrov E, Karpova O, Rodionova N. Potato virus X: structure, disassembly and reconstitution. Mol Plant Pathol 2007;8(5):667–75.

[11] Riechmann JL, Laín S, García JA. Highlights and prospects of potyvirus molecular biology. J Gen Virol 1992;73( Pt 1):1–16.

[1.09] Study of the fluorescence intensity decay of nanophotosensitizers using time-resolved spectroscopy methods

Fedor G. Bystrov¹, Vladimir I. Makarov² and Victor B. Loschenov^{1, 2}

¹National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Kashirskoe Shosse 31, Moscow, 115409, Russian Federation

²A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

E-mail contact: augustbystrov@gmail.com

Abstract: The effect of the biological environment on the fluorescence properties of aluminum phthalocyanine nanoparticles (nan-AlPc) was studied. The measurements were carried out using a detection system based on a Hamamatsu streak camera C10627 with picosecond temporal resolution, and a picosecond laser at a wavelength of 637 nm and 65 ps pulse duration. The presence of two fluorescence lifetimes, 5 ns and 10 ns, was registered for nan-AlPc incubated with macrophages. After photodynamic treatment a significant change in the fluorescence kinetics of nan-AlPc, deposited under mice skin autografts was observed. The obtained information on nan-AlPc fluorescence kinetics is fundamental for building a model of AlPc–biological environment interactions.

1 Introduction

Molecular photosensitizers (PS) are widely usеd in the treatment of cancer and inflammatory diseases. Despite the important and positive results achieved to date, there is a need to improve the selectivity of PS accumulation and a need to increase the selectivity of treatment impact. Taking into account these facts, the use of nanophotosensitizers is thought to be promising. Aluminum phthalocyanine nanoparticles are one of the representatives of this class of PS. In this work it could be shown that nan-AlPc can be used to assess the risk of skin autograft rejection, and that the application of colloidal solution of nan-AlPc on autografts leads to an increase of fluorescence intensity in the case of an inflammatory rejection [1]. Furthermore, our own studies have indicated a decrease of fluorescence intensity after prolonged exposure to laser radiation – the so-called photobleaching – that is absent for the molecular form of aluminum phthalocyanine. In this study the effect of the biological environment on the fluorescence properties of nan-AlPc was examined.

2 Materials and methods

A detection system was developed, consisting of a Hamamatsu streak camera C10627 with picosecond temporal resolution, a picosecond laser with 637 nm wavelength and 65 ps pulse duration, and a monochromator with fiber-optics input [2]. Research work was carried out in three steps. Based on the fact that the fluorescence of nan-AlPc injected in biological tissue appears mostly in pathologically changed areas, it was assumed that the pH of environment may affect the fluorescence. Thus, the first step was to study the fluorescence kinetics of nan-AlPc water colloids at different pH values. For this, five samples with pH values of 11, 10, 8, 3 and 2 were prepared and measured. It should be noted that nan-AlPc are insoluble in water and do not fluoresce under neutral pH conditions, which was confirmed by the sample with a pH=8.

The second step was to study the fluorescence kinetics of nan-AlPc incubated with immune cells. To simulate immune cells, the human monocytic leukemia cell line THP-1 was used. Monocytes were differentiated into macrophages using concanavalin A (ConA). Then samples with monocytes and with macrophages were incubated with nan-AlPc colloid. Thus, two samples were prepared: one with monocytes containing nan-AlPc and one with macrophages containing nan-AlPc.

In a third step, the possibilities of time-resolved measurements by means of the designed experimental system for in-vivo application were evaluated. For this, two mice with skin autografts were taken and nan-AlPc was added beneath one of the autografts. At first the intensity decay of nan-AlPc deposited under autografts was analyzed in order to identify the number of lifetime components and their distribution. Then experimental mice were exposed to photodynamic therapy (PDT) and were analyzed as before.

3 Results

The results obtained on the first, second and third steps of experiments are shown in Tables 1 and 2.

The differences in fluorescence kinetics for mouse #1 can be explained by taking into account the fact of formation of purulence under the skin autograft. For mouse #2, engraftment occurred normally.

4 Conclusion

Based on the fact that fluorescence of nan-AlPc is susceptible to the photobleaching effect that is absent in the molecular form of aluminum phthalocyanine, it can be assumed that the appearance of nan-AlPc fluorescence in the diseased tissue is not only due to the normal dissolution, but also due to the transition of surface molecules of the nanoparticle to the different orientation relative to the surface of nanoparticle caused by the peculiarities of the environment. The appearance of several fluorescence lifetime components, each of which corresponds to a particular state of the molecule on the surface, might be evidence of these transitions.

References

[1] Vasilchenko SY, Volkova AI, Ryabova AV, Loschenov VB, Konov VI, Mamedov AA, Kuzmin SG, Lukyanets EA. Application of aluminum phthalocyanine nanoparticles for fluorescent diagnostics in dentistry and skin autotransplantology. J Biophotonics 2010;3(5–6):336–46.

[2] Bystrov FG, Makarov VI, Pominova DV, Ryabova AV, Loschenov VB. Analysis of photoluminescence decay kinetics of aluminum phthalocyanine nanoparticles interacting with immune cells. Biomed Photon 2016;5(1):3–8.

[1.10] Spectral fluorescence method of bacteriochlorin accumulation dynamic estimation in mice skin with superficial wound Staphylococcus infection

Ekaterina V. Akhlyustina¹, Yulia S. Maklygina², Alexander V. Borodkin², Anastasia V. Ryabova^{1, 2}, Anastasia A. Kuneva³, Polina A. Rybakova³, Dmitry V. Yakovlev³, Gennady A. Meerovich² and Elena V. Filonenko⁴

¹National Research Nuclear University (NRNU), Moscow Engineering Physics Institute (MEPhI), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

²A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

³Pirogov Russian National Research Medical University (RNRMU), Ostrovitianov str. 1, Moscow, 117997, Russian Federation

⁴P. A. Herzen Moscow Cancer Research Institute – Branch of the National Medical Research Radiological Centre of the Ministry of Health of the Russian Federation, 2nd Botkinskiy proezd 3, 125284 Moscow, Russian Federation

E-mail contact: katya_ahlyustina@mail.ru

Abstract: Derivatives of bacteriochlorins (Bch) are promising for use as photosensitizers (PS) in the near-infrared (NIR) spectral range (750–850 nm) not only for photodynamic therapy (PDT) of tumors but also for the therapy of local infections in damaged tissue (especially septic inflammation) and other pathological processes. In a murine experimental model of a superficial wound Staphylococcus infection, it could be shown that the developed fluorescence spectroscopic approaches are promising to study the pharmacokinetics and biodistribution of PS based on Bch.

1 Introduction

The absorption of biological tissue in the NIR spectral region is minimal, which provides the possibility for effective PDT with Bch derivatives on sizable pathological processes [1]. Many photoactive substances are based on Bch which is hydrophobic and already used for PDT. Different nanostructured forms for intravenous injection such as liposomal, micellar, сremophor and cyclodextrin dispersions are created [2]. Bacteriochlorin derivatives have tumor affinity and effectively communicate with bacteria depending on their chemical composition, structure and dosage [3]. PS based on Bch have a rapid pharmacokinetic. That’s why the development of high-speed control methods for Bch biodistribution and pharmacokinetics measurement in vivo is important.

2 Materials and methods

A water cyclodextrin suspension of Bch in a concentration of 0.2 g/l was received from A. F. Mironov (Lomonosov Moscow State University of Fine Chemical Technologies). The Bch biodistribution after intravenous injection was studied in experiments on superficial wound Staphylococcus infection areas on Balb/c mice (males weighing 20–29 g). The skin of the mice backs had been previously epilated. Under anesthesia, a skin flap from the back was removed, and this place was seeded with the Staphylococcus culture. Two days later Bch was administered. The Bch dose amounted to 10 and 20 mg/kg of animal body weight.

For quantitative fluorescence measurements, the fiber-optic spectrometer LESA-01-BIOSPEC (BioSpec, Moscow, Russia) was used [4]. The fluorescence was excited with a 532-nm laser. The fluorescence value was calculated as the area under the fluorescence peak of the biological tissue between 800 and 880 nm (S_fl) divided by the area under the laser scattered peak of the biological tissue between 525 and 540 nm (S_l) – the so-called normalized fluorescence intensity (NFI):

This technique allows elimination of the laser instability, and partly takes into account the fluorescence reabsorption when quantifying the fluorescence intensity. The fluorescence video system (BioSpec, Moscow, Russia) was used simultaneously, with the fluorescence spectral measurements. The fluorescence video system operates for recording the fluorescence signal in a narrow wavelength range (820–870 nm) and obtaining the intensity in arbitrary units. The excitation wavelength used in the experiments was 532 nm.

3 Results

The proposed approaches allowed the Bch distribution to be examined at different times after administration with high spatial, spectral and temporal resolution. It could be demonstrated that the maximum concentration of Bch in the skin is achieved about 60–130 min after PS administration. Therefore, this time period is the most effective for PDT. The fluorescence intensity of Bch in the superficial wound Staphylococcus infection was higher than in normal skin (Figure 1). The average ratio of Bch fluorescence intensity in the wound versus skin surface was 11.8 for a Bch dose of 10 mg/kg and 21.8 for a Bch dose of 20 mg/kg. Thus the contrast was improved with an increase of Bch dosage from 10 to 20 mg/kg. The distribution of fluorescence intensity of the mice skin was obtained by the fluorescence video system at various time points after Bch administration (Figures 2A and B). Simultaneous two-dimensional control of the intensity distribution in the different mice body parts allowed for evaluation of the selectivity of the Bch accumulation dynamics (Figure 2C).

Figure 1:

Fluorescence spectra of Bch in the superficial wound infection area and in normal skin 120 min after injection.

Figure 2:

The fluorescence intensity of Bch. (A) In the superficial wound infection area (black-and-white shot), (B) in the superficial wound infection area (color shot), and (C) in organs post mortem.

4 Conclusion

Studies of Bch show that the spectral properties correspond to the maximum optical transparency region of biological tissues in the NIR giving rise to the suggestion that Bch could be a promising PS. The research results show that the developed fluorescence spectroscopic approaches are suitable to study pharmacokinetics and biodistribution of PS based on Bch, especially in the NIR and infrared regions.

Acknowledgment: This work was supported by Russian Foundation for Basic Research (Grant number: ‘15-29-04869-ofi-m’).

References

[1] Dai T, Huang YY, Hamblin MR. Photodynamic therapy for localized infections – State of the art. Photodiagnosis Photodyn Ther 2009;6(3–4):170–88.

[2] Mironov AF, Green MA, Tsiprovsky AG, et al. Photosensitiser based on bacteriochlorin p derivative, method of obtaining bacteriochlorin p derivative and method of photodynamic therapy of cancer with application of said photosensitiser. Patent RU2411943. http://russianpatents.com/patent/241/2411943.html [Accessed on September 16, 2016].

[3] Lukyanets EA, Makarova EA, Yakubovskaya RI, et al. Photosensitisers for photodynamic therapy. Patent RU2476218. http://russianpatents.com/patent/247/2476218.html [Accessed on September 16, 2016].

[4] BioSpec. Laser electronic spectrum analyzer for fluorescent diagnostics and photodynamic therapy monitoring. http://www.biospec.ru/2/LESA_e.pdf [Accessed on September 23, 2016].

[1.11] Study of aluminum phthalocyanine nanoparticle fluorescence properties changes in tissue engraftment for small laboratory animals cross skin transplantation

Dina S. Farrakhova¹, Ekaterina V. Akhlyustina¹, Vladimir I. Makarov², Daria V. Pominova¹ and Anastasia V. Ryabova^{1, 2}

¹National Research Nuclear University (NRNU), Moscow Engineering Physics Institute (MEPhI), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

²A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

E-mail contact: farrakhova.dina@mail.ru

Abstract: The possibility of aluminum phthalocyanine nanoparticles (nAlPc) application for evaluation of skin engraftment was studied and the fluorescence properties dynamics of tissue engraftment for cross skin transplantation of small laboratory animals were analyzed.

1 Introdution

Currently, one important research topic is the development of a skin condition evaluation method based on the luminescence spectral analysis of biological tissues. In the present study, a nAlPc colloidal solution was used as a photosensitizer. The spectroscopic skin properties analysis in the monitoring mode serves as a basis for an inflammation stage evaluation method for skin transplantation. When nAlPc get into inflamed biological tissue, they start exhibiting fluorescent properties. The fluorescence intensity increases with the rise of macrophage concentration in the inflamed tissue [1]. This method offers an opportunity to determine the inflammation stage and gives accurate prediction of transplanted engraftment evaluation.

2 Materials and method

The study was conducted on laboratory mice. Cross skin transplantation was realized on two skin areas of the mices’ backs. The nAlPc colloidal solution was added under the right autograft, and the left autograft was used as a control.

Skin autofluorescence and nAlPc fluorescence spectra analyses were conducted for evaluation of the interaction of nAlPc with biological tissues. The spectra were obtained using the spectroscopy system LESA-01-BIOSPEC (BioSpec, Moscow, Russia). A helium-neon laser (632.8 nm) was used for fluorescence excitation. Fluorescence spectra of autografts and normal skin were measured at 30 min; 1, 2.5, 3, and 4 h; and 1, 2, 4 and 7 days after surgery.

3 Results and discussion

The fluorescence spectra and the fluorescence intensity dynamics of autograft with nAlPc are presented in Figure 1.

Figure 1:

Fluorescence measurements. (A) The fluorescence spectra of autograft with nAlPc (mouse #1). (B) The graph of fluorescence intensity dependence on time for autografts and normal skin after surgery (mouse #1).

Fluorescence intensity increased with time, which indicates the increase of the inflammatory reaction after cross skin transplantation. As a consequence, the quality of tissue engraftment can be tracked with the help of nAlPc colloidal solution. This way allows action to be taken before the appearance of irreversible processes.

The nAlPc colloidal solution does not fluoresce which can be explained by fluorescence quenching in the molecular crystalline structure [2]. But nAlPc molecules begin to fluoresce when they interact with inflamed tissue. This fact is connected to a large concentration of macrophages in injured skin [1].

4 Conclusion

An evaluation was conducted of fluorescence properties dynamics of nAlPc in the tissue engraftment for cross skin transplantation of small laboratory animals. The nAlPc fluorescence intensity increases over time with the rise of macrophage concentration in the injured skin. This method allows non-invasive determination of the physiological condition of the skin surface and the evaluation of the stage and rate of engraftment or abruption of skin autografts. In addition, nAlPc colloidal solution may allow the acceleration of the healing process of autografts, because nAlPc can block an inflammatory response.

References

[1] Vasilchenko SY, Volkova AI, Ryabova AV, Loschenov VB, Konov VI, Mamedov AA, Kuzmin SG, Lukyanets EA. Application of aluminum phthalocyanine nanoparticles for fluorescent diagnostics in dentistry and skin autotransplantology. J Biophotonics 2010;3(5–6):336–46.

[2] Steiner R, Breymayer J, Rück A, Loshchenov V, Ryabova A. Crystalline organic nanoparticles for diagnosis and PDT. Proc SPIE 2015;9308:93080R.

[2.01] Femtosecond laser surgery of mammalian embryo and oocytes

A. A. Osychenko¹, A. A. Astafiev¹, A. M. Shakhov¹, A. D. Zalessky¹, A. A. Titov¹ and V. A. Nadtochenko^{1, 2, 3}

¹N. N. Semenov Institute of Chemical Physics of RAS, Kosygina str. 4, Moscow, 117977, Russian Federation

²Institute of Problems of Chemical Physics of RAS, Academician Semenov Avenue 1, Chernogolovka, Moscow region, 142432, Russian Federation

³Chemistry Department, Moscow State University, Moscow, 11999, Russian Federation

E-mail contact:nadtochenko@gmail.com

Abstract: Study of the size and dynamics of cavitation bubbles produced by focused femtosecond laser pulse and when the laser irradiation is focused on different components (organelles) in the mammalian oocyte is presented.

1 Introduction

Femtosecond laser nanosurgery of cells and embryos is a topical field of biophotonics. Currently, the mechanism of femtosecond laser irradiation absorption by embryo cells is not well understood. The most commonly used model of laser absorption in the cell is water because cells consist of more than 90% of water. But sufficient differences of chemical composition and properties between water and living cells limit the application of this model. Fully-grown mammalian oocytes (or germinal vesicle), rather than typical nucleoli when imaged, contain prominent, but structurally homogenous optical bodies called “nucleolus-like bodies” (NLBs). Figure 1 shows the enlarged nucleus of an oocyte before it develops into an ovum. The following components can be differentiated: (i) zona pellucida, the strong glycoprotein membrane surrounding the plasma membrane of mammalian oocytes, the layer that covers the oocyte, (ii) cytoplasm containing the cytosol, organelles, cytoskeleton, and various particles, (iii) germinal vesicle, and (iv) NLB immersed in germinal vesicle.

Figure 1:

Image of germinal vesicle oocyte in a phosphate buffer droplet. NLB – nucleolus-like body, GV – nucleoplasm in the germinal vesicle, CYT – cytoplasm, ZP – zona pellucida. The red and blue spots were irradiated by femtosecond laser pulses.

NLBs accumulate a vast amount of material, but their biochemical composition and functions remain uncertain [1]. The objectives of the present work are to: 1) study the size and dynamics of cavitation bubbles produced by focused femtosecond laser pulses when targeting different components (organelles) in the cell and 2) the examination of the development of oocyte after laser pulses were focused on the different organelles in the cell. Moreover, the probability to reach metaphase II (MII) stage of oocyte after laser surgery pulses directed to the particular organelles was determined.

2 Materials and methods

A titanium-sapphire (Ti:Sa) oscillator (Tsunami^®, Spectra-Physics) generated femtosecond pulses with a wavelength of 790 nm, a repetition rate of 80 MHz and a pulse energy close to 30 nJ. The Pockels cell (Avesta Project Ltd.) selected single pulses. Single pulses were amplified by Ti:Sa oscillator (TiF-20, Avesta Project Ltd.) amplifier. The amplified pulse energy provided 150 nJ. A laser attenuator consisting of a half-wave plate and a polarizing cube attenuated the pulse energy. The inverted optical microscope Olympus IX71 was used with a lens 40× 0.75NA (UPlanFLN, Olympus). The spot in the focal plane is characterized by the radius of the laser beam waist w₀=0.61λ/NA=0.64 µm, and a Rayleigh length of Z_R=1.63 µm. The formation of the cavitation bubbles was monitored by the measurements of the intensity of the backscattered probe light from the bubbles. The wavelength of the probe light was 445 nm. Light intensity was measured by a photomultiplier tube and LeCroy WaveSurfer 62Xs oscilloscope. The time resolution of the scattered light measurements was 5 ns. The Ethics Committee approved the experimental protocols of the work carried out at the Institute of Chemical Physics of RAS. The germinal vesicle oocytes were obtained from 4–8 week old C57BL/6J mice.

3 Results and discussion

It was found that when NLB material was irradiated the development probability of germinal vesicle oocytes to the MII stage is reduced by 3 to 7 times as compared with control measurements. This suggests the important role of the organization NLB material in the maturation of mouse oocytes. Laser shots directed to NLB, nucleoplasm in the germinal vesicle or cytoplasm does not effect on the probability of germinal vesicle oocyte to reach MII phase.

It was found, that both the size and lifetime of a cavitation bubble are significantly different during laser irradiation on the various organelles. Moreover, the bubble lifetime is different for different areas inside the cytoplasm. For example, for shots on the red dots it was 1.3 µs whereas on the blue dots it was 0.4 µs. It suggests meaningful heterogeneity of the cell and that the lifetime of the bubble can provide information about this heterogeneity.

Acknowledment: This work was supported by the Ministry of Education and Science of Russian Federation (Grant number: ‘14.604.21.0058’, unique identifier: ‘RFMEFI60414X0058’).

Reference

[1] Gulin A, Nadtochenko V, Astafiev A, Pogorelova V, Rtimi S, Pogorelov A. Correlating microscopy techniques and ToF-SIMS analysis of fully grown mammalian oocytes. Analyst 2016;141(13):4121–9.

[2.02] Optical tweezer on the base of 4-channel liquid crystal (LC) modulator for trapping of biological objects

Alexander V. Korobtsov¹, Svetlana P. Kotova^{1, 2}, Nikolay N. Losevsky¹, Aleksandra M. Mayorova^{1, 2} and Sergey A. Samagin¹

¹Lebedev Physical Institute, 221 Novo-Sadovaya Str., Samara, 443011, Russian Federation

²Samara National Research University, 34 Moskovskoye shosse, Samara, 443086, Russian Federation

E-mail contact: mayorovaal@gmail.com

Abstract: Laser tweezers proved to be of great importance for various applications in biology and medicine as a non-invasive sterile tool. They are used extensively in many biotechnical laboratories. The minimization of the negative laser radiation effect on trapped objects is one of the most critical problems during optical manipulation of micro-objects of biological origin to be investigated.

1 Introduction

One way to minimize negative laser radiation effects on trapped objects is the choice of the right radiation wavelength [1]. On the other hand, during trapping of eukaryotic cells with sizes significantly exceeding the size of point optical traps, the maximum of radiation is typically directed to the nucleus – the most optically dense structure of the cell [2]. Thus the stable trapping of cells by their peripheral part with traps in the form of rings, ellipses, and their arcs is the relevant problem.

For the formation of the contour traps, the use of a 4-channel liquid crystal (LC) modulator (LC focuser) is proposed. Its use in the scheme of optical tweezers allows generating optical traps in the form of rings and ellipses, and also controlling their size and shape in real-time by changing potentials of the control contacts.

2 LC focuser

Sufficient energy efficiency, a wider spectral range compared to that of a commercial multipixel spatial modulator, the simplicity and compactness of the device and the control system, and as a result lower cost of the scheme are the advantages of the LC focuser.

The scheme of the LC focuser is presented in Figure 1. Two modular cylindrical LC lenses are combined into one device. Nematic LC layer is sandwiched between two glass substrates covered with a transparent, high-resistance coating and low-resistance, non-transparent strip-shaped contacts.

Figure 1:

Scheme of liquid crystal (LC) focuser. 1 – glass substrates, 2 – contact electrodes, 3 – high resistive conductive layer, 4 – orienting coating, 5 – LC layer, 6 – spacers.

By controlling the electrical parameters of the device it is possible to change the voltage distribution on the aperture. The LC molecules become reoriented under the voltage applied (S-effect). This results in the change of the spatial distribution of the phase delay introduced by the LC layer into the transmitted light. Two operation regimes of LC focuser can be selected: (1) with small and (2) with high modal parameters. The physical meaning of this value is that the square of the modular parameter is the ratio of the resistance of the high resistive conductive layer and the LC layer impedance, and its magnitude determines the nature of the voltage distribution over the aperture.

To achieve an operation regime with small modal parameters, it is necessary to decrease the frequency and/or resistance of the transparent conductive coatings. In this regime, the voltage distribution and accordingly the shape of the profile of the phase delays are determined by the amplitudes and relative phases of the potentials. Thus, equipotential elliptic lines can be realized. These potential distributions are transformed to corresponding phase profiles in the form of elliptical or circular truncated cones. In the region of Fresnel diffraction, at small distances from the LC focuser, the points with maximum intensity in a transverse plane are located on the contour curve, replicated the shape of the equipotential lines of the voltage profile. Thus, one can generate the light fields with intensity distributions in the form of rings, ellipses (with the different relation of principal axes and their arbitrary orientation to aperture boundaries) and their arcs. Embedding the LC focuser into an optical tweezer scheme (Figure 2) it is possible to form optical traps of specific shapes.

Figure 2:

Scheme of optical tweezer with liquid crystal focuser.

3 Experiments

Trapping experiments were carried out using a wavelength of 532 nm. Saccharomyces cerevisiae yeast cells, suspended in water, were used as objects of biological origin for micromanipulation. The yeast cells are weakly absorbing objects, at the employed radiation wavelength. The optical traps were ring-shaped or elliptic with the capability to change their shapes and sizes. The trapping efficiency was estimated by the so-called escape velocities – the maximal velocities of the object stage motion for which the micro-object remained to be captured by the optical trap. This value depends on both the trap parameters and the type and size of micro-objects.

4 Results

It could be demonstrated that the contoured optical traps generated with the LC focuser effectively captured micro-objects which sizes were comparable to the optical trap sizes. Thus, the observed escape velocities of yeast cells (size of about 5×6 µm) were approximately 15 µm/s at 5.6 mW radiation power in the optical trap. The fragments of the video record, illustrating the capture and confinement of an individual yeast cell by the ring optical trap are presented in Figure 3.

Figure 3:

Capture and confinement of a single Saccharomyces cerevisiae cell. The arrows show the direction of the microscope stage movement.

It could be also shown that such optical traps can be used for the trapping of large (about 20 µm) micro particles. The yeast cells, aggregated into a single particle, were used as a large micro-object. Selected frames of the video of the experiment are presented in Figure 4.

Figure 4:

Trapping and confinement of a large microparticle. The arrows show the direction of the microscope stage movement.

In further experiments the dependence of the trapping efficiency on the optical trap shape was demonstrated as well as the possibility of controlling the trapping efficiency by changing the trap diameter employing ring traps. A decrease of the traps’ diameters resulted in an increase of the escape velocity at first but reached a constant afterwards. Due to this, it is possible to effectively trap micro-objects without affecting the objects’ central volume.

Numerical simulation showed that light fields with a transverse intensity distribution not only in the form of rings or ellipses but also in the form of squares, diamonds, parallelograms, and octagons could be generated using a LC focuser in operation regime with high modal parameter.

4 Conclusion

Taking into account the diversity of shapes and sizes of biological objects, and experimentally demonstrating the dependence of capture efficiency on optical trap shapes we believe that contour traps of such forms can also be employed in biomedical applications.

Acknowledgment: The work was supported by the Russian Foundation for Basic Research (Grant number: ‘16-29-14012’).

References

[1] Ashkin A, Dziedzic JM, Yamane T. Optical trapping and manipulation of single cells using infrared laser beams. Nature 1987;330(6150):769–71.

[2] Fore S, Chan J, Taylor D, Huser T. Raman spectroscopy of individual monocytes reveals that single-beam optical trapping of mononuclear cells occurs by their nucleus. J Opt 2011;13(4):44021.

[2.03] After-effect of low-intensity of HeNe laser irradiation on the activation of ATP synthesis and reprogramming of the genome

Tiina I. Karu, Valentina M. Manteifel and Ludmila V. Pyatibrat

Institute of Laser and Information Technologies of RAS, Pionerskaya St. 2, Troitsk 142092, Moscow Region, Russian Federation

E-mail contact: tiinakaru47@gmail.com

Abstract: The effects of low-intensity laser radiation (LILI) on the structure of mitochondria were studied. Changes within the mitochondria indicate activation of oxidative phosphorylation, which may be the result of genome reprogramming.

1 State of research

The effect of LILI on the structure of mitochondria is being investigated. Mitochondrial changes reflect the activation of oxidative phosphorylation, which may be the result of genome reprogramming. Exposure of human peripheral blood lymphocytes in vitro by a helium neon laser (at a radiant exposure of 56 J/m²) resulted in the fusion of 80% of initially separate mitochondria (there are about 40 small mitochondria in normal cells). One hour after irradiation the cells contain 2–4 elongated branched mitochondria and very few small mitochondria [1]. The total volume of mitochondria per cell did not change.

Fusions of the bulk of mitochondria usually indicate an increased adenosine triphosphate (ATP) synthesis via oxidative phosphorylation. It is known that such changes require the presence of specific proteins responsible for fusion of the outer mitochondrial membranes [2]. The synthesis of these proteins is controlled by the expression of the corresponding genes (МFN1 mitofusin-1, МFN2 mitofusin-2, OPA1).

Laser irradiation has an after-effect on mitochondria in successive generations of yeast Torulopsis sphaerica. Delivery of 460 J/m² radiant exposure results in changing giant mitochondrion in the cells cultivated for 18 h [3]. First, the mitochondrial matrix is expanded and then the relative proportion of cristae membranes is increased. It is known that cristae membranes contain the protein complexes required for the mitochondrial electron transport chain (ETC). The detected changes of mitochondria may reflect the acceleration of ATP synthesis. The increasing of the cristae per mitochondrion can be due to changed expression of genes regulating the energy metabolism. For example, exposure of fibroblasts by LILI led to an increased expression of genes, encoding sub-units of the protein complexes in the ETC [4, 5].

The hypothesis that the target of LILI is the cytochrome c-oxidase is now supported experimentally [6]. Absorption of photons by this enzyme results in accelerating electrons flow in the ETC; this causes an increase in ΔΨm and ATPm. Such functional changes have been identified a few minutes after irradiation.

2 Conclusion

Presented research findings indicate the effect of LILI on prolonged activation of the oxidative phosphorylation in the cells-descendants. Light-sensitive reorganization of the mitochondria may be regulated on the genetic expression level.

References

[1] Bakeeva LE, Manteifel VM, Karu TI. Effect of yeast Torulopsis sphaerica cell irradiation on the ultrastructure of the mitochondrial apparatus in descendant cells. Tsitologiya 1999;41(11):966–73.

[2] Hoppins S, Lackner L, Nunnari J. The machines that divide and fuse mitochondria. Annu Rev Biochem 2007;76:751–80.

[3] Manteifel V, Karu T. Rearrangements of mitochondrial ultrastructure in descendants of yeast cells irradiated with He-Ne lasers. Trends Photochem Photobiol 2011;12:77–91.

[4] Masha RT, Houreld NN, Abrahamse H. Low-intensity laser irradiation at 660 nm stimulates transcription of genes involved in the electron transport chain. Photomed Laser Surg 2013;31(2):47–53.

[5] Zhang Y, Song S, Fong CC, Tsang CH, Yang Z, Yang M. cDNA microarray analysis of gene expression profiles in human fibroblast cells irradiated with red light. J Invest Dermatol 2003;120(5):849–57.

[6] Karu TI. Mitochondrial signaling in mammalian cells activated by red and near-IR radiation. Photochem Photobiol 2008;84(5):1091–9.

[2.04] Laser impact monitoring during photocoagulation using optoacoustic technique

Anton Lytkin¹, Andrey Larichev¹, Svetlana Shmeleva¹, Varvara Simonova¹, Vladimir Sipliviy², Andrey Bolshunov² and Alesya Ardamakova²

¹Lomonosov Moscow State University, Leninskie Gory, Moscow, 119991, Russian Federation

²State Research Institute of Eye Diseases of the Russian Academy of Sciences, Rossolimo street, Moscow, 119021, Russian Federation

E-mail contact:aplytkin@gmail.com

Abstract: Here within a method is presented aiming at controlling the temperature during laser coagulation. The method is based on an optoacoustic technique that includes experimental determination of the laser light’s absorption coefficient and numerical calculations. Values for a series of chorioretinal samples ex vivo were obtained in the range of 1300–12,000 m⁻¹. A three-dimensional model of chorioretinal thermal heating was developed.

1 Introduction

Laser technology is a widespread phenomenon in ophthalmology. One of the most popular applications is laser coagulation. However, there is no reliable, rapid method for monitoring laser impact during operation available. The problem during laser coagulation is to avoid damage to the photoreceptor layer. A short pulse scanning laser is able to produce an acoustic pressure wave in the biological tissue that can be registered by an acoustic receiver. One widely known way to use optoacoustics (OA) is based on continuous pulsing [1, 2]. Another implementation of OA for remote laser impact control is presented in this work.

2 Experimental set-up and methods

The main components of the used set-up are the probe laser DTL-319QT (wavelength, 527 nm; pulse duration, 10 ns) and the cuvette. The cuvette has been constructed in a manner to simulate the eye structure. A biological sample, here the chorioretinal complex ex vivo, is placed on the stand in the cuvette, and the cuvette space is filled with saline solution to prevent tissue drying. The probe-laser pulses applied to the sample generate the acoustic waves. A lens focuses the laser beam onto the sample. The typical signal that was obtained in the experiment is shown in Figure 1. The absorption coefficient is determined from the acoustic waveform registered by the transducer and viewed by an oscilloscope. To simulate therapeutic laser heating a thermoregulation module with Peltier element was used.

Figure 1:

Typical acoustic wave obtained in the experiment. 1 – The main wave pulse contains information about the absorption coefficient. 2 – The reflected pulse is separated from the main pulse; it doesn’t interfere. 3 – Part of the curve is used to estimate the absorption coefficient.

3 Results

The first method, using an approximation, allowed to estimate the absorption coefficient of the sample tissues. Values for seven different series of experiments were obtained in the range of 1300–12,000 m⁻¹. The difference of the estimated value of the absorption coefficient within one series does not exceed 20%. The approximation equation is as follows:

where α is the absorption coefficient. Retinal pigment epithelium (RPE) is a thin 6-µm narrow layer surrounded by the vitreous body from one side and choroid from the other side. RPE is the only layer that is able to absorb laser irradiation propagating through the tissues. The laser beam has a Gauss-shaped form with 100 µm radius. Tissues that are far enough from the beam do not suffer from any heating, so the border conditions are T=0 for borders of the box [0×L_x; 0×L_y; 0×L_z] (here and below T means difference between native temperature T₀=37°C and final temperature). The mathematical equations for this model are presented as follows:

F=F(x,y,z) determinates the geometry of the system, γ is the angle between the beam and a normal to the RPE layer, χ=1.52×10⁻⁷ m²/s the coefficient of thermal conductivity, ρ=993 kg/m³ the density, C=4180 J/kg K the heat capacity and I₀ the intensity of the beam centre. x_c, y_c, and z_c are coordinates of the lesion center. An example of simulation result predicted for preset parameters is shown in Figure 2.

Figure 2:

Results of numerical calculations for therapeutic laser impact with an exposure time t=100 µs, a laser power N=100 mW, and a tissue absorption coefficient of 90,000 m⁻¹.

4 Conclusion

A method using a two-step approach for the determination of the laser impact during laser photocoagulation is presented. The first step involves the experimental determination of the laser absorption coefficient in eye tissues. Experimental determination using the OA technique was carried out for seven samples of the human chorioretinal complex. The obtained values lie within the range of 1300–12,000 m⁻¹. The difference of the estimated absorption coefficient within one series is not greater than 20%.

The second step requires absorption coefficient to be known and uses a simulation model to calculate the therapeutic laser impact.

References

[1] Brinkmann R, Koinzer S, Schlott K, Ptaszynski L, Bever M, Baade A, Luft S, Miura Y, Roider J, Birngruber R. Real-time temperature determination during retinal photocoagulation on patients. J Biomed Opt 2012;17(6):061219.

[2] Schlott K, Koinzer S, Ptaszynski L, Bever M, Baade A, Roider J, Birngruber R, Brinkmann R. Automatic temperature controlled retinal photocoagulation. J Biomed Opt 2012;17(6):061223.

[2.05] Joint application of fluorescence imaging and local fluorescence spectroscopy for photodynamic diagnostics and photodynamic therapy of skin cancer

Albert E. Mukhin¹, Alexander V. Borodkin², Pavel V. Grachev² and Eugene F. Stranadko³

¹National Research Nuclear University (NRNU), Moscow Engineering Physics Institute (MEPhI), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

²Laser Biospectroscopy Laboratory, A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

³State Science Center of Laser Medicine, Federal Medical and Biological Agency of the Russian Federation (FMBA of Russia), Studencheskaya st. 40, Moscow, 121165, Russian Federation

E-mail contact:mukhin.albert@gmail.com

Abstract: In this paper results of photodiagnostic studies on a patient with skin cancer of the ear are presented. A fiber-optic spectrometer LESA-01-BIOSPEC (BioSpec, Moscow, Russia) and a fluorescence video system were used for fluorescence spectra analysis and fluorescence imaging to determine the dynamics of photosensitizer accumulation over 3 h after drug injection before, during, and after photodynamic therapy (PDT).

1 Introduction

According to data of incidence of malignancies and mortality from 2014 in the Russian territories, skin is a leading localization for cancer incidence (12.6 %; with melanoma 14.2%). Vast amount of current research is devoted to PDT of oncological diseases, including malignant skin tumors. The undoubted advantage of PDT is the possibility of combining the treatment itself and the fluorescence diagnostics, also known as photodiagnosis (PD) in a single procedure. In the case of skin cancer, PD is the most promising diagnostic method to identify the borders of tumor proliferation, since the sensitivity of this method is much higher than other modern methods of diagnosis.

2 Materials and methods

A patient with squamous skin cancer on the ear was admitted to the Department of Oncology, State Science Center of Laser Medicine. To conduct therapy and diagnostics chlorin e6 (Ce6) was used as photosensitizer. Ce6 is approved for the medical use in Russia [1] as “FOTODITAZIN” (LLC “VETA-GRAND”, registration number LS-001246, since 18.05.2012), and was administered intravenously at a dose of 1 mg/kg body weight.

PD was conducted before, during and after PDT utilizing two different types of equipment. A laser spectral system LESA-01-BIOSPEC was used for fluorescence spectral analysis (fluorescence excitation wavelength, 632.8 nm). For spectral detection the ear was visually divided into 5 areas (Figure 1A), in each of which fluorescence spectra were recorded throughout the therapy. Additionally a dual-channel laparoscopic fluorescence video system for visual evaluation of the photosensitizer accumulation in the normal and cancerous areas was employed. The system was operated in real-time mode throughout the therapy (excitation using 635 nm).

Figure 1:

Photodiagnosis. (A) The ear of the patient with highlighted areas: section 1 – upper part of auricle, section 2 – the back of auricle, section 3 – over the section 4, section 4 – over the “recess” behind the auricle, section 5 – under the “recess” behind the auricle. (B) Fluorescence video system images after administration of the photosensitizer. The cancerous areas are imaged in green.

The irradiation was carried out by the LFT-02-BIOSPEC laser system (BioSpec, Moscow, Russia) with a PDT activation wavelength of 662 nm and an irradiance of 270 J/cm². A sapphire needle capillary was used at the end of therapy making it necessary to obtain a weaker irradiance to remain in the pain tolerance of the patient.

3 Results

PD performed 3 h after drug injection using fluorescence video imaging of the tumor allowed the border of proliferation and accumulation of the photosensitizer to be demarcated. Visualization by means of the black-white diagnostic and color navigation cameras of the used fluorescence video system clearly showed the boundary between cancerous and normal areas (Figure 1B).

PD by the laser spectral system before phototherapy revealed a high level of photosensitizer accumulation in the cancerous areas relative to the normal skin (on the neck); the average fluorescence index was 1:2.2. During therapy and after irradiation, the dynamics of accumulation changed: in all areas it decreased due to photobleaching; particularly in section 2 the fluorescence index fell more than 2 times. The spectra after therapy showed a rise of the photosensitizer’s accumulation in all areas possibly due to an increased blood flow.

4 Conclusion

The data obtained by a joint application of fluorescence imaging and local fluorescence spectroscopy indicate a high efficiency of fluorescence diagnostics method of skin cancer and show the importance of photobleaching tracking of the chlorin series photosensitizer during irradiation.

References

[1] Stranadko EF. Main stages of development of photodynamic therapy in Russia. Biomed Photonics 2015;4(1):3–10.

[2.06] Light fields in skin tissue with rough surface

A. P. Ivanov and V. V. Barun

B. I. Stepanov Institute of Physics, NASB, 68 Nezavisimosti Ave., Minsk, 220072, Belarus

E-mail contact:ivanovap@dragon.bas-net.by and barun@dragon.bas-net.by

Abstract: The fluence rate inside skin tissue and its diffuse reflectance are analytically simulated. The roughness of the skin surface and light refraction at the epidermis and stratum corneum interface are taken into account. Light penetration depth is shown to be independent of the skin relief, whereas the reflectance increases with roughness variance.

1 Introduction

Light characteristics in- and outside biotissues provide the basis for solving various biomedical optics problems, such as the optimization of light therapy, laser hypothermia, and optical diagnostics methods. Irradiating and scattered light interacts with the rough skin surface, experiencing a small change in its power and angular distribution parameters. This has effects on the spatial distribution, the absorbed and scattered power, the light penetration depth, and the action spectra of tissue chromophores. Variations of refractive indices at the tissue layers’ interfaces result in changes of the angular patterns near the surface and the depth. Therefore, it is interesting to evaluate the influence of these factors on the light fields in skin tissue and on the retrieval of its parameters.

2 Tissue model and simulation method

The investigations are based on the optical and structural tissue model [1] and the evaluation [2] of skin roughness parameters. The tissue is treated as a three-layer medium comprising optically thin stratum corneum and epidermis and optically thick dermis. The model includes the set of thicknesses d_sc and d_e of the thin layers, volume fractions f_m and C_V of melanin and capillaries, and blood oxygen saturation S. In our investigations the following constants were assumed: d_sc=20 µm, d_e=100 µm, C_V=0.02, and S=0.75. The skin surface relief is simulated by the Gaussian probability density of micro area tilts, with variance D_γ ranging from 0 (plane Fresnel surface) to 0.44.

Analytic approaches [3, 4] were used to solve the radiative transfer equation in a multi-layered absorbing and scattering medium for the simulations. Multiple scattering and multiple light re-reflections between the skin layers and the surface are considered. Light angular patterns near the surface are set as:

where θ is the polar angle, η(θ)=asin(nsin θ/n_i), with n=1.55 and n_i=1.33 as the refractive indices of stratum corneum and epidermis (the same for dermis), respectively. These three angular functions describe light propagation in the corresponding tissue layers. Angle η treats the refraction of light propagating from the dermis (n_i) to surface (n). As a first step, the diffuse reflectance as a function of η and D_γ [5], and the angularly integrated surface albedo R^* is calculated for a surface irradiation and by the light scattered in the tissue volume. Thus the problem of obtaining the light characteristics in- and outside the tissue, reduces to the calculation of the light fields for a medium with a surface albedo depending on its skin roughness. This can be achieved using the method as described by Barun and Ivanov [4].

3 Results

In the first calculation step the surface albedo R^* was determined as a function of variance (D_γ), which behaves differently according to the wavelength λ. In the red, R^* decreases with increasing D_γ. On the other hand, the albedo R^* has a maximum in the blue with small variances D_γ ≈0.07–0.08, where R^* of a rough surface increases by approximately 1.3–1.5 times compared to a plane interface. The reasons are discussed in detail elsewhere [5]. Naturally, one can observe the corresponding opposite features in light transmission (equal to 1–R^*) for a rough skin surface. These features lead to spectral dependences of a tissue’s diffuse reflectance R_sk shown in Figure 1. In the blue, one can see that the albedo values depend slightly on the skin roughness. The opposite situation occurs in the red and near-infrared, where R_sk increases absolutely by 0.05 to 0.1, and the respective relative values do the same by 25 to 50%. This is due to the higher transmission of diffuse, multiply-scattered light, by a rough skin surface as compared to a plane one. Figure 1 illustrates the calculations made for the plane surface without accounting for the refraction. This factor is seen as dominantly affecting the spectral albedos R_sk.

Figure 1:

Skin albedo spectra at f_m=0.16 (solid lines, ●) and 0.04 (dashed, ■), symbols ● and ■ show calculations without accounting for the refraction, D_γ=0 (curves 1, ●, ■), 0.2 (2), and 0.44 (3).

Another important parameter of the light fields in a turbid medium is the light’s propagation depth. It determines, for example, light action on tissue chromophores, tissue heating by light, etc. A definite conclusion on this parameter can be obtained from the data shown in Figure 2. It illustrates the depth structure of the scattered fluence rate W at 800 nm, deep inside skin tissue. At the upper dermis region, W values are reduced by the influence of skin roughness and angular pattern I(θ). Here the essential effect of light refraction on W can also be noted. However, in deeper tissue regions at larger z, neither the roughness nor the refraction affects the fluence rate. It follows from these data, in particular, that light penetration depth is essentially independent of the two factors studied in the paper.

Figure 2:

Fluence rate W as a function of tissue depth z at f_m=0.16 (solid lines, ●) and 0.04 (dashed, ■), ● and ■ shows calculations without refraction, D_γ=0 (1, ●, ■) and 0.44 (2), λ=800 nm.

4 Conclusion

The above presented results enable answering the question, when one should account for the roughness of skin surface and light refraction while solving direct and inverse problems of biomedical optics. As pointed out, neither the roughness nor the refraction affects the fluence rate at large z. Another situation occurs for studies of tissue heating by red or near-infrared radiation. For example, it is shown in [6] that the main heating mechanism here is heat transfer from the epidermis to the dermis. One can see from Figure 2 that the roughness and the refraction result in the reduction of the fluence rate in the epidermis. So this layer would have a lower temperature, which reduces dermis heating too.

There are various known techniques to retrieve structural and biophysical parameters of soft tissues by using backscattered light. For example, the method to non-invasively determine f_m, d_e, C_V, and S via the spectral albedo R_sk is proposed in [7]. It follows unambiguously from the above results that the solution to the inverse problem requires accounting for the skin roughness and the angular pattern I(θ). However, the solution is complicated by the dependence of reflectance R^* on the parameters desired. It is favorable in this respect that our simulations show a slight effect of C_V and S on R_sk, so that one can set I_d(η)≡1. Function I(θ) will only depend on the product f_m·d_e that can be retrieved [7] by measuring R_sk at two wavelengths λ=500 and 570 nm. Besides, the proposed calculation model enables one to determine the variance D_γ which can be useful for evaluations of the efficiency of various dermatologic and cosmetic procedures. The similar remarks can also be applied to other methods of retrieving tissue parameters such as spatially resolved spectroscopy.

References

[1] Barun VV, Ivanov AP. Thermal effects of a short light pulse on biological tissues. I. An optical-thermophysical model. Biofizika 2004;49:1004–12.

[2] Barun VV, Ivanov AP. Light scattering by a rough surface of human skin. 1. The luminance factor of reflected light. Quantum Electron 2012;43(8):768–76.

[3] Zege EP, Ivanov AP, Katsev Il. Image transfer through a scattering medium. Berlin: Springer-Verlag;1986.

[4] Barun VV, Ivanov AP. Light absorption in blood during low-intensity laser irradiation of skin. Quantum Electron 2010;40(4):371–6.

[5] Barun VV, Ivanov AP. Light scattering by rough surface of human skin. II. Diffuse reflectance. Quantum Electron 2013;43(10):979–87.

[2.07] Photodynamic therapy of gonarthrosis with Fotoditazin

Tatyana A. Zharova¹, Sergei V. Ivannikov¹, Alexey M. Tonenkov¹, Evgueni F. Stranadko², Lyudmila A. Semenova³, Michael M. Smorchkov⁴, Vladimir I. Makarov⁵, Igor D. Romanishkin⁵, Anastasia V. Ryabova^{5, 6} and Victor B. Loschenov^{5, 6}

¹I. M. Sechenov First Moscow State Medical University, Trubetskaya st. 8-2, Moscow, 119991, Russian Federation

²State Science Center of Laser Medicine, Federal Medical and Biological Agency of the Russian Federation (FMBA of Russia), Studencheskaya st. 40, Moscow, 121165, Russian Federation

³V. A. Nasonova Scientific Research Institute of Rheumatology, Russian Academy of Medical Sciences, Kashirskoye Highway 34A, Moscow, 115522, Russian Federation

⁴N. N. Priorov Central Research Institute of Traumatology and Orthopedics, Russian Federation Ministry of Health, Priorov st. 10, Moscow, 127299, Russian Federation

⁵A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

⁶National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Kashirskoe Shosse 31, Moscow, 115409, Russian Federation

E-mail contact:zharova-mma@yandex.ru

Abstract: Specific features of chlorin e6 (Ce6) accumulation in tissues of a knee joint and efficiency of photodynamic therapy (PDT) for gonarthritis treatment were studied in an animal model. Fluorescence diagnostics in knee joint tissues with Ce6 can be used in clinical practice at gonarthritis before, during and after PDT for monitoring of Ce6 accumulation and treatment control.

1 Introduction

Degenerative-dystrophic changes of joints represent themselves as one of the numerous and severe groups of locomotory diseases. Osteoarthrosis is a group of diseases of different etiology that are based on causing harm to all of the joint components (cartilage, subchondral bone, synovial membrane, capsule, ligaments, periarticular tissue).

2 Materials and research methods

The experimental research conducted on 35 rabbits-males (Chinchilla breed) pertains to the application of a post-traumatic gonarthritis model. Specific features of Ce6 derivatives photosensitizer (PS) accumulation in tissues of a knee joint and efficiency of PDT for gonarthritis treatment were studied experimentally. Fluorescence spectroscopy revealed that the maximum Ce6 accumulation in the synovial membrane of a rabbit’s damaged knee joint is 2.5 h after its intravenous injection (Ce6 dose of 1.25 mg/kg). PDT was carried out with the application of laser irradiation at 662 nm wavelength using an intra-joint irradiation method. The target area was equal to 1.0 cm², and an energy density of 240–300 J/cm² was delivered to the joint tissues.

3 Results

Ce6 accumulation dynamics in the inflamed tissue in various compartments of a knee joint reveal that the maximum concentration of Ce6 is registered 2.5 h after administration.

Fluorescence diagnostics in knee joint tissues with Ce6 can be used in clinical practice at gonarthritis before, during and after PDT for monitoring of Ce6 accumulation and treatment control. The optimal radiation energy density was determined as 150 J/cm². In the studied time intervals (5–25 min) no dependency of the PDT effect on the irradiation time at an identical energy density was observed.

4 Conclusion

Chlorin series PS is promising and can be used to treat gonarthritis in clinics.

[2.08] Simulation of thermographic IR images of a localized heat source hidden in biological tissue

A. P. Ivanov and V. V. Barun

B. I. Stepanov Institute of Physics, NASB, 68 Nezavisimosti Ave., Minsk, 220072, Belarus

E-mail contact:ivanovap@dragon.bas-net.by and barun@dragon.bas-net.by

Abstract: Thermal imager data are simulated at varying power, depth, and dimensions of an internal heat source. The main idea of research is to get insight into tissue depth by using observations of tissue surface. The observed quantities are discussed as applied to various inverse problems of source parameters retrieval.

1 Introduction

Currently thermographic methods on the base of infrared (IR) images are actively incorporated into medical practice. These methods are based on the non-invasive recording of thermal images of open biotissue surfaces in order to identify, for example, an inflammatory pathology in internal regions of a studied organ having an enhanced temperature, or a dystrophic pathology with a reduced temperature. In spite of obvious benefits of these methods, they are not widely applied in medicine. One of the main limiting factors is that the thermal image recording from a tissue surface only indirectly bear information on the temperature regime of an internal region that is shadowed by intermediate tissue layers. The effects of biotissues on the results cannot, naturally, be excluded. In other words, the thermal action of a tissue slab between the investigated tissue region and the thermal imager creates noisy images. Because of this, techniques to correct the IR images for their refinement and for the extraction of information on the temperature of the interested tissue area are much needed. Such techniques would enable one to make conclusions on parameters of the thermal regime of internal organs and to derive their temperatures by a non-invasive way without temperature sensors implanted into the tissue. The design of the correction method will enable one to qualitatively improve the information content and the validity of diagnostic techniques for practical physicians and provide a powerful tool for studying a person’s organism by scientists. This paper discusses the IR imaging of biotissue surfaces, gives relations between tissue parameters and characteristics of an internal heat source, and outlines means to solve the inverse problem on retrieving the source characteristics from image data of a thermal imager.

2 Simulation method

As a basis for the investigations, the known stationary solutions [1, 2] for spatial temperature distributions created in a semi-infinite medium by elementary heat sources, namely by point spherical and line cylindrical ones, under Newtonian heat exchange at the surface are used. By integration over spatial coordinates, these solutions can be obviously generalized for a spherical or cylindrical source of an arbitrary radius. So, one can compute the temperature in any point of a medium. A thermal imager records usually IR radiation from an object, not its temperature directly. Each volume element of biotissues radiates according to the Planck’s formula and Kirchhoff’s law. Owing to the non-uniform spatial temperature distribution, one needs to integrate over tissue depth to get an optical signal (or radiance) from the exiting surface at any of its point. While integrating, the extinction of light should be accounted for. Here within a wavelength range of about λ=2–10 µm is considered. Due to the strong water absorption, let the extinction be provided by radiation absorption only with scattering neglecting. By doing so, the temperature and IR radiation images are computed.

3 Errors in temperature measurements owing to volumetric glowing of biotissue

It is well known that the skin temperature is usually lower than that of a tissue volume. Therefore, a thermal imager observes not the tissue surface temperature, but some higher value due to the volumetric glowing. Figure 1 illustrates the effect of systematic error δT₀ in measured surface temperature on the estimate of source temperature excess ΔT_s. Here solid lines are theoretically calculated, and dashed ones are re-calculated on the basis of tissue temperature with accounting for the volumetric tissue glowing. Consider the effect of the glowing due to a heat source on the error in retrieving the temperature excess ΔT_s of the latter. Let the experimentally measured value of ΔТ correspond to point A of Figure 1. Then, according to line 4, temperature ΔT_s corresponding to point A^* will be determined. The ΔТ value should be really decreased by δT₀, i.e. to go to point B, which corresponds to ΔT_s of point B^*. In this case, the error δТ_s in ΔT_s will be about 1 K. In other words, the volumetric tissue glowing provides for an enhanced source temperature, which can be important for solving the respective inverse problems.

Figure 1:

ΔТ versus ΔТ_s. Curves 1, 2, and 3 – h=0.2 cm⁻¹; curves 4, 5, and 6 – h=0.8 cm⁻¹; curves 1 and 4 – monochrome detector; curves 2 and 5 – spectral PtSi/Si_1-xGe_x detector; curves 3 and 6 – spectral PtSi/Si detector.

4 On the solution to inverse problems

Illustrate some opportunities of retrieving various characteristics of tissue and of internal heat source by using thermographic IR images. Let one observe the surface temperature rise created by a spherical heat source at several radial coordinates r from the source center. Then the theoretical nomograms of relative temperature rise shown in Figure 2 opens the opportunity to retrieve simultaneously the source depth, a, and the heat exchange parameter, h. The relative rise means here the temperature increase at point r with respect to that at r=0. When thermophysical characteristics of tissue and source depth are known, one can determine the Q/κ value, where Q is the heat power (in W/cm³) and κ, is the biotissue thermal conductivity [in W/(cm K)], by using theoretical equations for measured surface temperature rise dependence ΔT(r,0). Note the appreciable effect of parameter h on the temperature.

Figure 2:

Nomograms for retrieving simultaneously source depth a and heat exchange parameter h. Numbers near thin and bold curves give a (in cm) and h (in cm⁻¹) values, respectively.

5 Conclusion

Recording a thermal image of a person’s body finds currently wide applications in medical practice, e.g. for diagnosing organism conditions, or exposing pathological body regions, etc. Usually, the gathered image data are transformed into surface temperature maps. But there, obviously, arise a number of questions. For example, what a value is measured, if the temperature distribution over the body depth is non-uniform? This paper gives a quantitative answer to this question. Namely, the estimates are provided here to relate data of a thermal imager with thermophysical, optical and geometrical parameters of a system under consideration. Note that this system includes both the depth-varying temperatures of biotissue under the action of a human organism and spatially varying ones under the action of a heat source. The latter can simulate a pathological tissue region. The second question occurs from the following physically transparent fact. Really, one observes a tissue surface, but desires to retrieve a temperature of an internal heated region. How are the observations related with a quantity of interest? Here analytical tools are provided to solve the inverse biooptical problem on deriving not only the source temperature, but also the heat source depth, its thermal power and dimensions, thermophysical parameters of tissue itself, etc. The presented data can be used for designing dedicated algorithms and program codes of IR biomedical image processing for various medical and biophysical applications.

References

[1] Carslaw HS, Jaeger JC. Conduction of heat in solids. Second edition. Oxford: Clarendon Press; 1959.

[2] Draper JW, Boag JW. The calculation of skin temperature distributions in thermography. Phys Med Biol 1971;16(2):201–11.

[2.09] Laser systems and fiber optic tools for photodynamic therapy

Kirill G. Linkov¹, Vladimir V. Volkov¹ and Irina A. Shikunova²

¹A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

²Institute of Solid State Physics of RAS, 2 Academician Ossipyan str., Chernogolovka, Moscow District, 142432, Russian Federation

E-mail contact:rkir@mail.ru

Abstract: New therapeutic laser systems and fiber-optic light delivery tools were designed for further development of fluorescence diagnosis (FD) and photodynamic therapy (PDT) methods. Features and advantages of the developed laser equipment and possible applications of the fiber-optic instruments are considered.

1 Introduction

FD and PDT are promising new approaches in a wide spectrum of medical disciplines such as urology, dermatology, gynecology, gastroenterology, ophthalmology, and neurosurgery [1–4]. Since the 1990ies, laser equipment for FD and PDT has been developed at the Laser Biospectroscopy Laboratory of the A. M. Prokhorov General Physics Institute of the Russian Academy of Sciences and the designed equipment speeks for itself. From 1996–1998 many devices were transferred in the clinics, and since then are still working flawlessly. In total, clinics and academic institutions of Russia and some foreign countries obtained more than 300 units developed by our team. About a quarter of them are laser therapeutic devices.

An important part of equipment are fiber-optic systems. They are used both for diagnosis and therapy. At present, polymeric and quartz optical fibers are the most acceptable fibers in medicine for light delivery systems. Ordinary light scattering fiber tips, such as frontal or cylindrical ones have undoubtedly found a wide-spread medical application for PDT in routine clinical practice. To date, there are a number of light scattering fiber tips using different manufacturing methods. They are intended for clinical use, for example, for illumination of skin diseases, subcutaneous tumors of hollow organs and for the treatment of brain tumors or prostate.

2 Laser therapeutic systems

Advanced laser therapeutic systems have been developed. Development was carried out with the maximum addition of modern electronic components, taking into account the current requirements for optimizing costs and import substitution. List of modifications of the systems is repeatedly expanded, including versions with different emission wavelengths and output optical power. It is possible to manufacture laser devices for use with photosensitizers registered in Russian Federation and promising photosensitizers being developed or tested currently. Regulatory requirements for medical devices, published at the time of development, were taken into account. The focus has been on improving the reliability of the equipment in order to ensure a long-term and trouble-free operation.

A new medical laser therapeutic system was registered, has passed all necessary tests and was delivered in the clinic in 2016 under the model designation “Laser System LPhT-02-BIOSPEC”.

3 Fiber-optic tools for therapy

Regarding the therapeutic light delivery systems, different applicator types were developed, which look like a thin capillary coupled with a quartz fiber [5]. Experimental ex-vivo and in-vivo studies revealed the ability of such applicators to provide different types of therapy, namely PDT, hyperthermia, interstitial laser coagulation and laser-induced interstitial thermotherapy. All these kinds of laser interstitial minimal invasive therapies permit selective photothermal tumor destruction with a protection of the surrounding organ structure. The expansion of the tissue-destroying effect is highly dependent on the laser parameters and applicator type involved.

As an example, results for the controlled interstitial treatment, from PDT to coagulation, on pig liver (ex vivo) using a fiber-optic tool with sapphire needle capillary could be presented. The main advantages of this method include the implementation of temperature stabilization for interstitial PDT and the ability to control laser power by feedback circuit using an infrared camera.

Figures 1 and 2 show the temperature distribution along the sapphire needle capillary during laser irradiation. The x-axis is the distance along the applicator length (in mm). The y-axis is the temperature (in °C). In PDT mode the temperature is controlled and maintained between 40 and 43°C to provide the most effective therapy. Temperature for coagulation mode is about 85°C.

Figure 1:

Photodynamic therapy mode. The chosen laser power is 2 W.

Figure 2:

Coagulation mode is added to photodynamic therapy. The chosen laser power is 5.7 W.

The method of photo or thermal impact by laser radiation on the lesions needs a subsequent preclinical trial both as alternative method of a tumor therapy for primary or recurrent cases, and combined with surgery.

References

[1] Belyaeva LA, Adamyan LV, Stepanyan AA, Linkov KG, Loshchenov VB, Laser fluorescence spectroscopy with 5-aminolevulinic acid in operative gynecology. Laser Phys 2004;14(9):1207–13.

[2] Zavodnov VY, Kuzin MI, Kharnas SS, Linkov KG, Loschenov VB, Stratonnikov AA, Posypanova AM. Palliative treatment of patients with malignant structures of esophagus. Proc SPIE 1996;2625:482. doi:10.1117/12.230999.

[3] Model SS, Savelieva TA, Linkov KG. System for determining the concentration and visualization of the spatial distribution of photosensitizers based on tetrapyrrole compounds in the tissues of the human ocular fundus. Proc SPIE 2013;8699:86990E. doi:10.1117/12.2016675.

[4] Savelieva TA, Loshchenov VB, Volkov VV, Linkov KG, Goryainov SA, Potapov AA. The method of intraoperative analysis of structural and metabolic changes in the area of tumor resection. Proc SPIE 2014;9129:91290T. doi: 10.1117/12.2052480.

[5] Shikunova IA, Volkov VV, Kurlov VN, Loshchenov VB. Sapphire needle capillaries for laser medicine. Bull Russ Acad Sci Phys 2009;73(10):1345–8.

[2.10] Non-invasive blood glucose monitoring with THz reflection spectroscopy

Olga P. Cherkasova¹, Maxim M.Nazarov^{2, 3} and Alexander P. Shkurinov^{2, 4}

¹Institute of Laser Physics of RAS, Siberian Branch, pr. Lavrentyeva 13/3, Novosibirsk, 630090, Russian Federation

²FSRC “Crystallography and Photonics” RAS, Str. Butlerova 17A, Moscow, 117342, Russian Federation

³National Research Centre “Kurchatov Institute”, Akademika Kurchatova pl. 1, Moscow, 123182, Russian Federation

⁴Lomonosov Moscow State University, Leninskie Gory, GSP-1, Moscow, 119991, Russian Federation

E-mail contact:o.p.cherkasova@gmail.com

Abstract: Human skin optical properties were studied in vivo using terahertz (THz) time-domain spectroscopy with silicon Dove prism in the attenuated total internal reflection (ATR) configuration. The measurements were carried out on volunteers with normal blood glucose concentration and after glucose intake. The variations of the reflection spectra of human skin were correlated with the changes in blood glucose level. Our results demonstrate the possibility of a non-invasive real-time measurement of blood glucose concentration.

1 Introduction

Monitoring of glycemic status is considered to be a cornerstone of diabetic patients’ care. Measurements of glucose concentration in blood are best determined by standardized laboratory techniques using blood plasma biochemistry analyzers. However, disadvantages are the long measurement time, and a relatively large sample volume needed to obtain results [1, 2]. During the last 20 years, many portable blood glucometers for humans have appeared on the market. They are readily available, inexpensive, and provide immediate results while utilizing small quantities of capillary blood [1, 3]. However, control of diabetes mellitus involves daily self-monitoring of blood glucose by finger puncture several times a day to obtain a blood sample for further analysis. This procedure is invasive, painful, non-safe and unpleasant for patients. In the past decades much attention had been paid to the development of spectroscopic methods for non-invasive glucose measuring. These approaches include polarimetry, near-infrared spectroscopy, Raman spectroscopy, photoascoustics and optical coherence tomography [4].

Terahertz time-domain spectroscopy (THz-TDS) has not yet found wide application in this field. A distinctive feature of this method is the possibility of measuring directly the refractive index, absorption coefficient, and hence complex permittivity spectrum of the sample in a single scan and in a broad frequency range. The application of THz spectroscopy for studies of both normal human skin and skin pathologies in vivo has been reported previously [5]. To our knowledge, there are no studies available investigating the human skin optical characteristics when glucose concentration in blood is varied. However, it has previously been shown that the transmission coefficient of animal ear skin in sub-THz (0.03–0.04) and THz (0.34) frequency ranges correlates with blood glucose concentration [6, 7]. In the present paper, studies on human skin using THz-TDS in vivo in the 0.2–2 THz range are described.

2 Materials and methods

ATR spectra of human skin at normal blood glucose concentration levels and their variations during a standard oral glucose tolerance test were measured. A THz time-domain spectrometer described previously [8, 9] was used. Experiments were carried out using ATR optical scheme with a silicon right angle Dove prism, with p-polarized radiation.

3 Results

The ATR spectra of human palm skin after ingesting glucose solution are shown in Figure 1A.

Figure 1:

(A) The ATR amplitude |Rp| of human skin at different times (0, 45, 90 min) after ingesting a glucose solution. (B) The ATR amplitude R_int of human skin and glucose concentration in blood (in mM) versus time (in min) after glucose intake.

A 0.2 ml 84% glycerol solution in distilled water was used to improve optical contact and to increase the THz field penetration depth into the skin. The processing time of glycerol was 10 min. Since the temporal shape of reflected THz pulse, E(t), is not changed considerably, the pulse amplitude as the integrated characteristics of ATR amplitude R_int (Figure 1B) might be used:

Here E_s indicates the signal, E_r the reference; E_max and E_min are the corresponding maximal and minimal values in the time-domain. The largest variations in the ATR spectra were observed for the 0.1–0.5 THz frequency range. The amplitude of ATR signal of human palm skin is changed when blood glucose concentrations rise above the normal levels.

Analysis of the experimental reflection spectra was performed by comparing the experimental skin spectra with the dielectric function of the skin model. The shape of THz spectra of the biological tissue is mainly determined by water present in the skin, having a strong dispersion at low frequencies. The dielectric permittivity of water can be described by the Debye model.

4 Conclusion

Human skin spectra in vivo using THz-TDS in ATR optical scheme were measured. The ATR spectra of palm skin were consecutively measured 0–90 min after glucose intake at standard oral glucose tolerance test. Glycerol was used to improve the optical contact between the palm skin and the surface of the prism and thus to increase the sensitivity of the method. The largest variations of the ATR spectra were observed within the 0.1–0.5 THz frequency range. These variations of the optical characteristics of human skin were correlated with the changes in blood glucose level. The ATR amplitude of human palm skin increased when the glucose concentrations in blood rose above the normal level. The changes observed in the spectra are described with good accuracy by the reduction in the ratio Δε₁/τ_D in the Debye model of the glucose aqueous solution. This change in the response of bound water is the reason of the sensitivity of in-vivo THz skin measurements to high glucose concentration in blood. The results demonstrate the possibility of non-invasive real-time measurement of blood glucose concentration.

Acknowledgment: This work has been supported by Russian Foundation for Basic Research (Grant numbers: ‘14-02-00846’, ‘14-02-00979’ and ‘16-52-00222’).

References

[1] Rebel A, Rice MA, Fahy BG. Accuracy of point-of-care glucose measurements. J Diabetes Sci Technol 2012;6(2):396–411.

[2] Summa NM, Eshar D, Lee-Chow B, Larrat S, Brown DC. Comparison of a human portable glucometer and an automated chemistry analyzer for measurement of blood glucose concentration in pet ferrets (Mustela putorius furo). Can Vet J 2014;55(9):865–9.

[3] Clarke SF, Foster JR. A history of blood glucose meters and their role in self-monitoring of diabetes mellitus. Br J Biomed Sci 2012;69(2):83–93.

[4] He R, Wei H, Gu H, Zhu Z, Zhang Y, Guo X, Cai T. Effects of optical clearing agents on noninvasive blood glucose monitoring with optical coherence tomography: a pilot study. J Biomed Opt 2012;17(10):101513.

[5] Zaytsev KI, Gavdush AA, Chernomyrdin NV, Yurchenko SO. Highly accurate in vivo terahertz spectroscopy of healthy skin: Variation of refractive index and absorption coefficient along the human body. IEEE Trans Terahertz Sci Technol 2015;5(5):817–27.

[6] Sigel PH, Lee Y, Pikov V. Millimeter-wave non-invasive monitoring of glucose in anesthetized rats. 39th IEEE International Conference on Infrared, Millimeter, and Terahertz waves (IRMMW-THz). 14–19 September 2014, Arizona, USA. doi: 10.1109/IRMMW-THz.2014.6956294.

[7] Sun C-K, Tsai Y-F, Chen H. Method and device for detecting a blood glucose level using a electromagnetic wave. United States Patent US 2013/0289370 A1. https://www.google.com/patents/US20130289370 [Accessed on September 24, 2016]

[8] Cherkasova OP, Nazarov MM, Angeluts AA, Shkurinov AP. The investigation of blood plasma in the THz frequency range. Opt Spectrosc 2016;120(1):55–63.

[9] Cherkasova OP, Nazarov MM, Berlovskaya EE, Angeluts AA, Makurenkov AM, Shkurinov AP. Studying human and animal skin optical properties by THz time-domain spectroscopy”, Bull Russ Acad Sci Phys 2016;80(4):479–83.

[2.11] On the estimation of tissue optical parameters from diffuse reflectance spectroscopy

Walter C. P. M. Blondel^{1, 2}, Prisca Rakotomanga^{1, 2}, Maria Kholodtsova^{1, 2, 3, 4}, Christian Daul^{1, 2}, Viktor B. Loschenov^{3, 4}, Marine Amouroux^{1, 2} and Charles Soussen^{1, 2}

¹Université de Lorraine, CRAN, 2 Avenue de la Forêt de Haye, 54516 Vandoeuvre-Lès-Nancy cedex, France

²CNRS, CRAN, 54516 Vandoeuvre-Lès-Nancy cedex, France

³A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

⁴National Research Nuclear University (NRNU), Moscow Engineering Physics Institute (MEPhI), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

E-mail contact:m.kholodtsova@gmail.com and loschenov@mail.ru

Abstract: This contribution is a state-of-the-art overview on inverse problem solving for spatially resolved diffuse reflectance spectroscopy (SRDRS) challenging the precise estimation of multi-layer biological tissue optical parameters.

1 Introduction

Spatially resolved (also called steady-state) diffuse reflectance spectroscopy is a fiber-optical spectroscopy technique studied in many clinical applications for about two decades, especially in cancerology, to diagnose pathological modifications in epithelial tissues of skin and hollow organs such as the oral cavity, upper respiratory tract, lung, bladder, upper and lower digestive tracts, and cervix. The system, shown in Figure 1, usually consists of a multiple fiber-probe with several source-detector separations (SDSs), spectrophotometer(s) and a broadband non-ionizing white light source (spectral range, 400–800 nm). SRDRS proved to be a valuable “optical biopsy” tool allowing clinicians to probe biological tissues non-invasively, helping them to improve pre-operative diagnostic and therapeutic guiding efficiency. Advantages of this approach are:

Figure 1:

Main constitutive parts of an SRDRS instrumentation set-up (left top), schematic correspondence of the multi-layer configuration between tissue, mathematical and physical simulation and model (left below) and general scheme of the inverse problem to be solved for estimating the optical properties of the tissue from SRDRS measurements (adapted from [9]) (right).

1. Rather simple and cost-effective instrumentation is needed which is favorable for clinical transfer.

2. Its resolution is not limited by the Abbe resolution.

3. It is highly sensitive to changes in the concentration of chromophores such as melanin or oxy- and deoxy-hemoglobin, the size of the scattering centers (for instance cell nucleus) and the structural organization of the medium (cell orientation, multiple layers).

It provides sets of spatially and spectrally resolved bulk reflectance data from which the local properties of the tissues are to be unmixed. Thus, the robust and precise estimation, at various tissue depths (see Figure 1), of the absorption and scattering coefficients (µ_a and µ_s) as well as the anisotropy factor (g) remains a challenging problem in the development of SRDRS in order to provide new key features for characterizing pathological changes in tissues [1–8]. Therefore, the present contribution aims at providing a systematic overview of the most recent contributions and latest advances on the estimation of the optical properties.

2 Description of the inverse problem to be solved

As shown in Figure 1, the inverse problem to be solved consists of in finding the parameters of the mathematical and/or physical model, namely p=[μa1..m(λ),μs1..m(λ),g1..m(λ),t1..m] (for a m-layer tissue model with thicknesses t^1..m), according to the data observed i.e. spatially resolved diffuse reflectance spectra. Because of the complexity of the interactions between light and biological tissues and the complexity of the tissue structure itself, this inverse problem is intrinsically ill-posed (close “similar” results may be obtained with different sets of parameter values) and ill-conditioned (the solutions may suffer from numerical instability due to finite precision and errors in the experimental and forward problem simulated data). Firstly, mathematical and numerical modelling of the physical phenomena of light–tissue interaction, together with a physical model of the tissue, have to be chosen to describe the forward problem appropriately. The latter provides a set of spatially resolved (r) spectra I_mod(λ, r; p) simulated with the tissue parameter vector p. Secondly, an objective function (also called energy- or cost-function) is defined in order to quantify the difference (referred as a “distance”) between the experimental and modelled data sets I_exp(λ, r) and I_mod(λ, r; p) respectively. Thirdly, an optimization procedure is applied with the aim of minimizing the latter distance by iteratively varying the values of the parameters in p. Thus, precise and robust convergence of the optimization procedure towards “true” (ground truth) values of the searched parameters is at stake throughout the choice of the cost-function and the optimization method.

3 Solutions proposed

Monte Carlo-based simulations are mainly used for modelling the forward problem as it is well appropriate to handle the complex geometry of the tissues, the complexity of the photophysical interactions and the geometrical features of the optical probe, especially for short SDSs. Solutions implementing fast graphics processing unit (GPU)-based and/or look-up table based approaches are commonly employed [1, 4–6, 9–12] but also multi-layer diffusion models [3, 13] are used. However, the inherent statistical noise in I_mod, which increases with SDSs, has to be taken into account and its impact on local minima occurrence risk to be properly evaluated. The convergence of the optimization procedure highly depends also on the form of the cost function, which can include spectral and/or spatial normalization in the data-term and sometimes a regularization term. Nonlinear least-squares curve fitting methods, such as the Levenberg–Marquardt algorithm, are most popular although the latter belongs to local minimum search approaches. In order to improve the convergence towards an unique global minimum solution, strategies are developed around genetic algorithms [9, 14]. The latter are also appropriate for handling the high dimension of the problem when taking into account the spectral resolution of the parameters to be estimated. Errors between ground truth known values of the parameters and their estimated values range from a few % up to several tens of % depending on the methods chosen among those mentioned above for every part of the problem.

Acknowledgments: This work was partly conducted in the frame of co-supervised PhD thesis between Université de Lorraine, CRAN-CNRS (France, Nancy) and A. M. Prokhorov General Physics Institute of RAS (Russian Federation, Moscow). The authors acknowledge the financial support for PhD grant of M. Kholodtsova from Conseil Régional de Lorraine and French Embassy in Moscow.

References

[1] Hennessy R, Markey MK, Tunnell JW. Impact of one-layer assumption on diffuse reflectance spectroscopy of skin. J Biomed Opt 2015;20(2):27001.

[2] Radosevich AJ, Eshein A, Nguyen TQ, Backman V. Subdiffusion reflectance spectroscopy to measure tissue ultrastructure and microvasculature: model and inverse algorithm. J Biomed Opt 2015;20(9):097002.

[3] Liao YK, Tseng SH. Reliable recovery of the optical properties of multi-layer turbid media by iteratively using a layered diffusion model at multiple source-detector separations. Biomed Opt Express 2014;5(3):975–89.

[4] Zhong X, Wen X, Zhu D. Lookup-table-based inverse model for human skin reflectance spectroscopy: two-layered Monte Carlo simulations and experiments. Opt Express 2014;22(2):1852–64.

[5] Fredriksson I, Larsson M, Strömberg T. Inverse Monte Carlo method in a multilayered tissue model for diffuse reflectance spectroscopy. J Biomed Opt 2012;17(4):047004.

[6] Liu C, Rajaram N, Vishwanath K, Jiang T, Palmer GM, Ramanujam N. Experimental validation of an inverse fluorescence Monte Carlo model to extract concentrations of metabolically relevant fluorophores from turbid phantoms and a murine tumor model. J Biomed Opt 2012;17(7):077012.

[7] Wilson RH, Mycek MA. Models of light propagation in human tissue applied to cancer diagnostics. Technol Cancer Res Treat 2011;10(2):121–34.

[8] Liu Q, Ramanujam N. Sequential estimation of optical properties of a two-layered epithelial tissue model from depth-resolved ultraviolet-visible diffuse reflectance spectra. Appl Opt 2006;45(19):4776–90.

[9] Kholodtsova MN, Daul C, Loschenov VB, Blondel WCPM. Spatially and spectrally resolved particle swarm optimization approach to increase precision in optical properties estimation by means of tissular diffuse-reflectance spectroscopy. Opt Express 2016;24(12):12682–700.

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[14] Jäger M, Foschum F, Kienle A. Application of multiple artificial neural networks for the determination of the optical properties of turbid media. J Biomed Opt 2013;18(5):57005.

[2.12] Near-infrared imaging for angiography in diabetic foot

Zera N. Abdulvapova¹, Pavel V. Grachev², Gagik R. Galstyan¹ and Olga N. Bondarenko¹

¹Endocrinology Research Centre, Dm. Yl’yanova Str. 11, Moscow, 117036, Russian Federation

²A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

E-mail contact:zera1987@mail.ru

Abstract: Current modern methods for lower limb ischemia (LLI) assessment have a number of limitations in diabetic patients. Indocyanine green (ICG) fluorescence angiography (ICGA) is a new technique for assessing the perfusion disturbance in LLI.

1 Introduction

Non-invasive assessment of lower limb circulation is a cornerstone in the evaluation of the severity of peripheral arterial disease. In clinical settings, ankle pressure (AP), toe pressure (TP), and indices derived from their comparisons with arm pressure, as well as transcutaneous oxygen tension (t_cpO₂) and skin perfusion pressure (SPP), are currently available for measuring lower limb hemodynamics. The first two parameters only reflect the severity of arterial occlusive disease and the latter two the local perfusion as well. In addition, t_cpO₂ and SPP may be measured at different, predetermined sites. Both of these methods, however, are limited in their applicability. Measurements can only be taken from the healthy area surrounding foot ulcers, and attachment of the probes is likely to be successful only on flat surfaces. Furthermore, the probes cannot be applied directly onto the ulcer [1]. ICGA is a new technique to assess the perfusion disturbance in ischemic limbs.

2 Materials and methods

This study was performed in four diabetic patients with ischemic limbs and foot ulcers. The principle of fluorescence imaging used in ICGA is simple: illuminating the tissue of interest with light at an excitation wavelength of 785 nm while observing it at longer emission wavelengths (810–850 nm). The device is equipped with a 785-nm light-emitting diode (LED) as an excitation light source and a charge-coupled device (CCD) camera covered with a lens and filter set. Real-time fluorescence images were displayed on a monitor and recorded using the digital image processing method of the audio video interleave (AVI) system. To evaluate the ICGA study, multiple parameters were obtained and analyzed to assess the perfusion. These parameters included time of ICG intravenous injection (T₀), onset of basal intensity (T_bas), onset to maximum intensity from T₀ (Tmax₀), onset to maximum intensity from T_bas (Tmax_bas), time from the onset to the end of intensity (Tend_bas) level of basal intensity (I_b), level of maximum intensity (Imax), the rate of intensity increase from baseline to peak intensity over time (IngR), the area under the curve of intensity over time (curve integral), the intensity at the end of the study (E_I), the magnitude of intensity decrease from peak intensity to the end of the study (E_n), and the rate of intensity decrease from peak intensity to the end of the study (EnR).

These parameters were assessed in several regions of interest (ROI): ROI 1, distal region of the first metatarsal bone; ROI 2, distal region of the forth metatarsal bone; ROI 3, the dorsum of the foot from Chopart’s joint to the Lisfranc joint; ROI 4, 5 cm below the medial epicondyle (Figure 1). Each region was 1.5×1.5 cm in size.

Figure 1:

Regions of interest for indocyanine green fluorescence angiography test.

3 Results

There were not any adverse reactions during ICGA procedure. Data from all ROIs with different ICGA parameters were collected (Figure 2). There were differences in Tmax₀ and Tmax_bas in the different ROIs, reaching more than 10 s. Ongoing studies with patient recruitment database and ICGA parameters are presented.

Figure 2:

ROIs of patient #1 and its parameters.

4 Conclusion

Foot wounds can be healed only with an appropriate level of blood supply and limb salvage efforts are highly dependent on perfusion to the lower limbs. ICGA provides rapid qualitative visual and quantitative information about regional foot perfusion. Future studies may include pre- and post-debridement and post-closure site tissue viability, pre- and post-vascular intervention levels of arterial flow, lower extremity flap viability, and comparisons to the transcutaneous partial pressure of oxygen test as well as other non-invasive hemodynamic measurements.

References

[1] Terasaki H, Inoue Y, Sugano N, Jibiki M, Kudo T, Lepäntalo M, Venermo M. A quantitative method for evaluating local perfusion using indocyanine green fluorescence imaging. Ann Vasc Surg 2013;27(8):1154–61.

[3.01] Sensitizer-nanoparticles for tissue diagnostics and photodynamic therapy

Rudolf Steiner^{1, 4}, Claudia Scalfi-Happ¹, Rainer Wittig¹, Anastasia Ryabova^{2, 4}, Susanna Gräfe³ and Victor B. Loschenov^{2, 4}

¹Institut für Lasertechnologien in der Medizin und Meßtechnik an der Universität Ulm, Helmholtzstr. 12, 89081 Ulm, Germany

²A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

³Biolitec Research GmbH, Otto-Schott-Str. 15, 07745 Jena, Germany

⁴National Research Nuclear University (NRNU), Moscow Engineering Physics Institute (MEPhI), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

E-mail contact: rudolf.steiner@ilm-ulm.de

Abstract: Nanoparticles of different kinds of materials are used to enhance tissue diagnostics or the efficacy of photodynamic treatment (PDT). However, approved substances are mostly molecular solutions of porphyrins, phthalocyanines or chlorins. However it is also possible to use nanoparticles (NPs) of the raw material of such organic compounds, which are not soluble in water and not fluorescent. In-vitro experiments with meta-tetra(hydroxyphenyl)chlorin (mTHPC) show that mainly macrophages will take up the NPs. Once inside the cells, molecules are dissolved from the NPs and are fluorescent and photoactive. Therefore, NPs of mTHPC can specifically be used for fluorescence diagnostics of inflamed or cancerous tissue and for PDT.

1 Basics

Nanoparticles made from aluminum phthalocyanine (AlPc) raw material, or mTHPC, are non-fluorescent because of fluorescence quenching due to the molecular crystalline structure forming a stack of flat molecular layers. However, when AlPc molecules become detached from the particles, fluorescence occurs. First observations demonstrated the benefit of using aluminum phthalocyanine nanoparticles (nAlPc) for the assessment of the rejection risk of skin autografts in mice by measuring fluorescence intensities of detached AlPc molecules. Skin autografts showing a high fluorescence intensity of AlPc were finally rejected, induced by an inflammatory process. In contrast, autografts with normal skin autofluorescence were accepted [1]. Therefore, nanoparticles or nano-emulsions from appropriate photosensitizers such as porphyrins, chlorins or phthalocyanines, offer a new promising drug delivery system for hydrophobic sensitizers [2, 3]. They can be used for fluorescence diagnostics and treatment by PDT [4]. However, the reaction process has to be evaluated.

2 Applications

Nanoparticles made from large-dispersed aluminum phthalocyanine, or mTHPC, crystals form a stable colloid suspension. The nanoparticles are therefore suitable for clinical use due to the possibility of good transportation in aqueous media and penetration into tissue. They are also suitable for fluorescence diagnostics because such nanoparticles do not initially fluoresce but when molecules dissolve from the nanoparticles, in the monomeric molecular form, fluorescence occur [1, 4]. These findings could be used to specifically detect inflammatory processes (Figure 1) or tumors and have the potential of using nAlPc, or mTHPC nanoparticles, as a new treatment modality for PDT.

Figure 1:

Fluorescence of tissue inflammation after macrophages have taken up mTHPC nanoparticles and molecules have dissolved from the nanoparticles inside the cells.

3 Our studies

In addition to fluorescence, spectroscopic and microscopic techniques, confocal Raman microspectroscopy has become a powerful tool for the investigation of living cells and biological samples [5]. In a previous study our research group investigated the role of lipids in the discrimination between Caco-2 colon carcinoma cell line and the rat intestine epithelial cell line IEC-6 by confocal Raman microscopy with the alpha300 R Raman microscope (WITec GmbH, Germany) described previously [6]. It might also be helpful to elucidate the fluorescent appearance of AlPc molecules after detachment from the nanoparticles, by taking the Raman-microspectroscopic approach to follow the cellular uptake of photosensitizer nanoparticles in their crystalline, non-fluorescent form (Figure 2). Therefore, crystalline nanoparticles of different size, made from hydrophobic porphyrin-derived photosensitizer, were applied to either L929 murine fibroblasts or to J774A.1 murine monocytes or macrophages and the results were compared with the properties of the sensitizer Foslip^®. In a further step, the dissolution process of the nanocrystals with increasing fluorescence signal was evaluated. These investigations can help to understand the effect of photosensitizer particle size on cellular uptake, the differences in internalization mechanisms of the studied cell lines, and the dissolution of photoactive molecules from the nanoparticles for diagnosis and PDT, taking also into consideration the reactions of different types of macrophages.

Figure 2:

Raman microscopic imaging of the chemical content of macrophages after uptake of nanoparticles to see intracellular reaction mechanisms.

References

[1] Vasilchenko SY, Volkova AI, Ryabova AV, Loschenov VB, Konov VI, Mamedov AA, Kuzmin SG, Lukyanets EA. Application of aluminum phthalocyanine nanoparticles for fluorescent diagnostics in dentistry and skin autotransplantology. J Biophotonics 2010;3(5–6):336–46.

[2] Lademann J, Richter H, Meinke MC, Lange-Asschenfeldt B, Antoniou C, Mak WC, Renneberg R, Sterry W, Patzelt A. Drug delivery with topically applied nanoparticles: science fiction or reality. Skin Pharmacol Physiol 2013;26(4–6):227–33.

[3] Ribeiro AP, Andrade MC, da Silva Jde F, Jorge JH, Primo FL, Tedesco AC, Pavarina AC. Photodynamic inactivation of planktonic cultures and biofilms of Candida albicans mediated by aluminum-chloride-phthalocyanine entrapped in nanoemulsions. Photochem Photobiol 2013;89(1):111–9.

[4] Zhang J, An F, Li Y, Zheng C, Yang Y, Zhang X, Zhang X. Simultaneous enhanced diagnosis and photodynamic therapy of photosensitizer-doped perylene nanoparticles via doping, fluorescence resonance energy transfer, and antenna effect. Chem Commun (Camb) 2013;49(73):8072–4.

[5] Scalfi-Happ C, Rück A, Udart M, Hauser C, Dürr C, Kriebel M. Label-free detection of tumor markers in a colon carcinoma tumor progression model by confocal Raman microspectroscopy. Proc SPIE 2013;8798:87980B-1.

[6] Scalfi-Happ C, Jauss A, Hollricher O, Fulda S, Hauser C, Steiner R, Rück A. Confocal Raman microscopy for investigation of the level of differentiation in living neuroblastoma tumor cells. Proc SPIE 2007;6630:66300T.

[3.02] Depth independent Cherenkov radiation-mediated therapy with 5-ALA photosensitizer

Yuliya S. Maklygina¹, Anastasia V. Ryabova^{1, 2}, Victor B. Loschenov^{1, 2}, Egor N. Sokolov³, Denis I. Nevzorov³, Elena Yu. Grigoreva³, Michail B. Dolgushin³ and Boris I. Dolgushin³

¹A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

²National Research Nuclear University (NRNU), Moscow Engineering Physics Institute (MEPhI), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

³N. N. Blokhin Russian Cancer Research Center of RAS, 24 Kashirskoe Shosse, Moscow, 115478, Russian Federation

E-mail contact: us.samsonova@physics.msu.ru

Abstract: The combination of light and photosensitizers (PS) for phototherapy is promising, but the shallow penetration of the laser radiation into tissue limits the method of photodynamic therapy (PDT). Therefore, one main goal of research work is to overcome these limitations by using Cherenkov radiation (CR) from radionuclides to activate the PS. Histological analysis of tumor sections has shown the selective destruction of cancerous cells (glioma C6). These results offer a way to achieve depth-independent CR-mediated therapy using different types of PS.

1 Introduction

Light-based methods suffer from the rapid attenuation of the light in tissue. A current assumption is that the CR could serve as a depth-independent light source for photoinduced therapy inside cancer tissue [1]. Radionuclides are an ideal source for CR because of their positron emission, which travel faster than the speed of light in the medium within the ultraviolet to visible spectrum (250–600 nm). Radionuclides such as fluorodeoxyglucose (¹⁸F-FDG) are widely used in positron emission tomography (PET) that allows the metabolic processes in the body to be observed, especially in clinical oncology. ¹⁸F-FDG is widely used to detect diverse tumors with exceptionally high sensitivity because it accumulates in highly proliferating tumor cells undergoing an enhanced glucose metabolism. As 5-aminolevulinic acid (5-ALA) has become an integral part in the treatment of malignant glioma, this PS was used for CR-induced therapy in the research presented here.

2 Materials and methods

In-vivo studies were performed on experimental animals with induced malignant glioma C6 in the groin. During the study intravenous injections of 5-ALA and tracer amounts of ¹⁸F-FDG were made successively at intervals of 2 h respectively. The 5-ALA acted as a PS whereas the ¹⁸F-FDG induced the CR; that in combination, resulted in CR-induced therapy.

Observation of metabolic processes was carried out and the ¹⁸F-FDG concentrations in tissues were imaged using PET. The evaluation of the photodynamic effect of CR-induced therapy was made using confocal laser scanning microscopy (CLSM).

3 Results

Histological analysis of tumor sections was visualized using CLSM post mortem. Analysis of tumor sections of 5-ALA-treated mice revealed proliferation of glioma C6 cells forming agglomerates (Figure 1A). Analysis of tumor sections of ¹⁸F-FDG + 5-ALA-treated mice revealed selective destruction of proliferating cells in the tumor region as well as pronounced necrotic zones that amounted to approximately 20–30% of the tumor mass (Figure 1B). Large areas of ¹⁸F-FDG + 5-ALA-treated tumors exhibited a loss of cellular architecture and a significantly higher distribution of apoptotic foci (Figure 1B). These findings suggest that the cell damage was probably mediated by the CR-induced free radicals. Thus a comparison of the ¹⁸F-FDG-untreated (Figure 1A) and ¹⁸F-FDG-treated (Figure 1B) tumor sections using CLSM shows predominantly apoptotic cells in the latter that confirms the selectivity of the method.

Figure 1:

Histological analysis of tumor sections of (A) 5-ALA-treated and ¹⁸F-FDG-untreated and (B) ¹⁸F-FDG + 5-ALA-treated cells by means of confocal laser scanning microscopy.

4 Conclusion

In this study, a new approach was demonstrated using CR from PET radionuclides to activate 5-ALA for phototherapy. The effect of the complimentary radical-generation mechanisms enabled an effective CR-induced therapy using tumor-targeted PS. Although ¹⁸F-FDG equally accumulates in non-tumor-associated pathologies, such as inflammation, CR-induced therapy is only effective when both the CR source and the PS are in the same cell, which minimizes off-target toxicity. The established biocompatibility of all the components used in the study creates a path to human translation. Thus the approach opens up the possibility of treating a variety of lesions by a depth-inside therapy.

References

[1] Kotagiri N, Sudlow GP, Akers WJ, Achilefu S. Breaking the depth dependency of phototherapy with Cerenkov radiation and low-radiance-responsive nanophotosensitizers. Nat Nanotechnol 2015;10(4):370–9.

[3.03] Development of methods for fluorescence imaging in theranostics for oncological diseases

Elena V. Filonenko¹, Andrey D. Kaprin¹, Antonia N. Urlova¹ and Maxim V. Loschenov²

¹National Medical Research Radiological Centre of the Ministry of Health of the Russian Federation, 10 Zhukov street, Obninsk, 249036, Kaluga region, Russia

²Laboratory of Laser Spectroscopy, A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

E-mail contact: derkul23@yandex.ru

Abstract: The work presented here describes a procedure for fluorescence imaging in theranostics for oncological diseases which includes visually assessed fluorescence diagnosis (FD), fluorescence spectroscopy, and fluorescence navigation.

1 Introduction

Detection of early cancer, intraoperative assessment of tumor borders and real-time control of efficacy of photodynamic therapy (PDT) represent very pressing medical problems. FD with photosensitizers has been used in the detection of early cancers and assessment of tumors on the surface of the skin and various mucosas [1, 2]. There are two main methods for the assessment of fluorescence imaging: visually and by values of diagnostic parameters derived from fluorescence spectroscopy. Visual assessment and fluorescence spectroscopy have their limitations due to the subjective nature of the interpretation of the color density of the images and the time-consuming calculation of diagnostic parameters. In this study, FD with 5-аminolevulinic acid (5-ALA) was used in patients with skin, laryngeal, and bladder cancer as well as lesions of the central nervous system and female reproductive organs.

2 Materials and methods

For FD, 5-ALA (Alasens) was given per os in the form of an aqueous solution at a dose of 20–30 mg/kg body weight 3 h before the procedure. Technical equipment produced by the companies Karl Storz (Tuttlingen, Germany) and Olympus (Tokio, Japan) was used for imaging under blue light excitation. A video-assisted fluorescent light emitting diode (LED) device UFF-630/675-01 (BioSpec, Moscow, Russia) was used during endoscopy. Fluorescence spectra in the range of 635–800 nm were recorded by means of a laser electron-spectrum analyzer (LESA-01-BIOSPEC, BioSpec, Moscow, Russia) with an emitting laser wavelength of 632.8 nm. During local fluorescence spectroscopy 5–65 spectra were recorded in every patient. The values of the diagnostic parameter (DP) were determined in all cases. The DP values were collected from several parts of the tumor under visual detection of fluorescence; then the tumor’s average value was calculated. DP was calculated automatically as the ratio of the peak area of protoporphyrin IX (PpIX) (690–720 nm) to the area of the reflected laser light (620–640 nm) indirectly representing the evidence of the Alasens-induced PpIX accumulation in the tissues. Fluorescence spectra of Alasens-induced PpIX in the intact tissue were recorded by determination of the fluorescence contrast of neoplastic/normal tissue. Fluorescence imaging for navigation in photosensitized tissues during PDT for malignant tumors in patients was performed with a novel device which consisted of two light sources: a LED-based white light source and another laser diode-based source for fluorescence excitation at 635 nm [3].

3 Results

For FD with visual evaluation of 5-ALA fluorescence (Figure 1), a sensitivity and specificity of 97% and 78% was observed, respectively. Complementing this method with local fluorescence spectroscopy (Figure 2) enabled increased specificity. Fluorescence navigation of photosensitized tumors enables detection of malignant tumors and their borders by visual assessment of the tumor and simultaneous collection of DP to perform treatment control on a real-time basis.

Figure 1:

Fluorescence diagnosis in a patient with bladder cancer.

Figure 2:

Fluorescence spectroscopy in a patient with gastric cancer.

References

[1] Filonenko EV, Sokolov VV, Chissov VI, Lukyanets EA, Vorozhtsov GN. Photodynamic therapy of early esophageal cancer. Photodiagnosis Photodyn Ther 2008;5(3):187–90.

[2] Filonenko EV. Fluorescence diagnostics and photodynamic therapy: justification of applications and opportunities in oncology. Biomed Photonics 2014;3(1)3–7

[3] Loshchenov M, Zelenkov P, Potapov A, Goryajnov S, Borodkin A. Endoscopic fluorescence visualization of 5-ALA photosensitized central nervous system tumors in the neural tissue transparency spectral range. Photonics Lasers Med 2014;3(2):159–70.

[3.04] Sapphire shaped crystals for phototheranostics and combined anticancer therapy

Irina A. Shikunova¹, Vladimir V. Volkov² and Vladimir N. Kurlov¹

¹Institute of Solid State Physics of RAS, 2 Academician Ossipyan str., Chernogolovka, Moscow District, 142432, Russian Federation

²A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

E-mail contact: sh_irina@issp.ac.ru

Abstract: Sapphire has a high refractive index and a broad transmission band spanning the ultraviolet, visible and infrared bands. Sapphire also has high hardness, very good thermal conductivity, tensile strength, and thermal shock resistance. The favorable combination of excellent optical and mechanical properties of sapphire, together with high chemical inertness and biocompatibility, high resistance to human blood and body fluids, makes it an attractive structural material for medical applications. We have developed new medical instruments and devices for combined laser photodynamic therapy and thermal therapy, laser surgery, fluorescence diagnostics, and cryosurgery based on sapphire crystals of various shapes with capillary channels in their volume grown by the EFG/Stepanov technique.

1 Sapphire smart scalpels

Sapphire smart scalpels, allowing simultaneous incision and fluorescence diagnostics of a resected tissue immediately during surgical operation, have been developed. The principle of this new kind of scalpel is based on the use of isolated capillary channels in the volume of the sapphire scalpel for the introduction of quartz waveguides (Figure 1). One of the waveguides is used for delivering the laser radiation directly to the narrow region of the cutting edge and for local excitation of photoluminescence. Another one is used for detection and transfer of the photoluminescence to a spectrometer. The scalpel employs rapid real-time feedback analysis for online diagnostics of tissues during surgery when the tumor is removed. A laser, optically coupled directly inside the edge, facilitates localization of the effective laser energy at the cutting edge of the scalpel, which is needed for hemostasis in the tissue adjacent to the incision [1, 2].

Figure 1:

Left: Sapphire ribbon with three capillary channels of 0.5 mm in diameter. Right: Sapphire scalpel used for diagnostics and coagulation of incised tissue during a surgical operation.

2 Sapphire needle capillaries

Sapphire needle capillaries were developed as new laser waveguide introducers for the delivery of laser radiation into a tumor during interstitial laser photodynamic therapy, thermotherapy and ablation of tumors (Figure 2). These needles allow one to increase the irradiation volume substantially, to obtain an optimal temperature distribution, to simplify the design, and to eliminate a system for cooling the device. The high degree of hardness of sapphire provides a stable point on the irradiator end for independent introduction of the irradiator into the tissue without using directors that lead to the increase in irradiator cross-section. The use of sapphire irradiators make it possible to improve the control over the dynamics of spatial photothermal distribution during the whole irradiation procedure, since the effective redistribution of released heat by the sapphire decreases the possibility of formation of overheating nuclei which can lead to the occurrence of thrombi, which are non-transparent to laser radiation [3].

Figure 2:

Left: Sapphire needle capillaries (outer diameter of 1.2 mm, inner diameter of 0.5 mm) in three different forms (top to bottom: as grown closed capillary, capillary with a point formed by mechanical operation of the rear and a capillary with a point and diffuser formed by mechanical operation). Right: Geometry of light of a sapphire needle (outer diameter of 1.2 mm, inner diameter of 0.5 mm) combined with a quartz fiber.

3 Sapphire multi-channel probe

In addition, a system was developed for the removal of brain tumors based on a sapphire multi-channel probe with demarcation of tumor borders by fluorescence diagnostics with simultaneous coagulation and aspiration (Figure 3). It allows for simultaneous laser coagulation for hemostasis, tumor aspiration through a channel, and local optical measurements of the properties of brain tissue for a more exact and complete removal of the malignant tissue [4].

Figure 3:

Left: Sapphire multi-channel probe for neurosurgery. Right: Neurosurgical operation for brain tumor removal using the sapphire probe.

4 Sapphire THz photonic crystal waveguides

The ability for broadband, low-loss terahertz (THz) waveguiding in sapphire multi-channel shaped crystals (Figure 4) could be also demonstrated. The waveguides have been characterized by both numerical simulations and experiments. They allow for guiding the THz waves in a multi-mode regime with a minimal power extinction coefficient of 2 dB/m at 1.45 THz [5]. These results demonstrate the capabilities of combining the advantages of EFG/Stepanov technology with the unique properties of sapphire (relatively low THz-wave absorption; high mechanical, thermal, chemical and radiation strength) for manufacturing the THz waveguides and endoscopic systems for medical applications.

Figure 4:

Sapphire THz photonic crystal waveguide.

References

[1] Kurlov VN, Rossolenko SN, Abrosimov NV, Lebbou K. Shaped crystal growth. In: Duffar T, editor. Crystal growth processes based on capillarity: Czochralski, floating zone, shaping and crucible techniques. New York: Wiley; 2010, p. 277–354.

[2] Shikunova IA, Volkov VV, Kurlov VN, Loshchenov VB. Sapphire needle capillaries for laser medicine. Bull Russ Acad Sci Phys 2009;73(10):1345–8.

[3] Kurlov VN, Shikunova IA, Ryabova AV, Loshchenov VB. Sapphire smart scalpel. Bull Russ Acad Sci Phys 2009;73(10):1341–4.

[4] Kiselev AM, Esin IV, Shikunova IA, Kurlov VN, Tereschenko SG, Lаpaeva LG. Combined neuronavigation in surgery of cerebral malignant tumor (only in Russian). Almanac Clin Med 2011;25:58–63. http://cyberleninka.ru/article/n/kombinirovannaya-neyronavigatsiya-v-hirurgii-zlokachestvennyh-opuholey-golovnogo-mozga [Accessed on September 19, 2016].

[5] Zaytsev KI, Katyba GM, Kurlov VN, Shikunova IA, Karasik VE, Yurchenko SO. Terahertz photonic crystal waveguides based on sapphire shaped crystals. IEEE Trans Terahertz Sci Technol 2016;6(4):576–82.

[4.01] Combined spectroscopic technique in low-grade glioma neurosurgery navigation

Tatiana A. Savelieva^{1, 2}, Sergey A. Goryaynov³ and Alexander A. Potapov³

¹A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

²National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

³N. N. Burdenko Neurosurgery Institute, 16 4th Tverskaya-Yamskaya Street, Moscow, 125047, Russian Federation

E-mail contact: savelevat@gmail.com

Abstract: A method for the simultaneous in-vivo analysis of fluorescence, scattering and absorption of brain tissues in adjacent spectral ranges from 500 to 800 nm is proposed. It helps to delineate low-grade glial tumors with low accumulation of fluorescent tumor marker by using other spectroscopic characteristics of tumor tissues.

1 Background

Fluorescence navigation during the resection of low-grade glioma (LGG) is not always a successful strategy. The reason for this lies in the limited accumulation of the fluorescent marker (as a rule 5-ALA induced protoporphyrin IX is used) in the tissues of benign gliomas. However, optical navigation techniques are extremely convenient for microsurgery. These two factors built the motivation for developing a method of combined analysis of multiple optical characteristics of low-grade glial tumors.

2 Materials and methods

In this report, a method is proposed for the simultaneous in-vivo analysis of fluorescence, scattering, and absorption properties of brain tissues in the spectral range of 500–800 nm. A detailed description of the device is given in [1]. The specific features of its application to LGG, particularly with regard to the structure and biochemistry of the tumors that affect the spectral signal, are described. The method was used in the N. N. Burdenko Neurosurgery Institute on 13 patients with LGG. The results of the spectroscopic analysis were confirmed by histological examination.

3 Results

The absence of a fluorescence signal was observed in six patients, while the other spectroscopic features of tumor changes were present.

One of the interesting results was a negative correlation (−0.75) between the fluorescence level (the ratio of the fluorescence signal to the intensity of the backscattered laser signal used as the fluorescence index) and the tissues’ light scattering properties (Figure 1A). This means that the optical density of tumor tissues is lower than normal tissue in case of low-grade glial tumors. The scattering signal from the center of the tumor was on average two times lower than that of the normal white matter.

Figure 1:

(A) Fluorescence index–scattering diagram registered spectroscopically in vivo for one patient with gemistocytic astrocytoma. (B) Oxygenation–scattering diagram registered spectroscopically in vivo for one patient with oligoastrocytoma.

A model of pathological changes, which explains this observation, is proposed. The tumor is characterized by an active growth, which leads to a high degree of vascularization of tissues and, consequently, improves blood supply to the tissue. This can be observed spectroscopically by an increase of the absorption coefficient. Thus there are some structural changes, namely, the displacement and the degradation of myelinated nerve fibers, a growth in the size and number of nuclei, and mitochondria degradation. All these changes have different effects on the optical scattering properties of tissue. Degradation of nerve fibers leads to a reduction of large scale scatterers in tissue as well as mitochondria degradation. At the same time increase of the size and number of nuclei has the opposite effect on the optical scattering coefficient. Thus, at various stages of tumor development, its scattering coefficient varies non-monotonically. But it is always lower than the scattering coefficient of normal tissues.

In our study, a positive correlation (0.43) between the level of tissue oxygenation and the degree of light scattering was observed (Figure 1B). This is due to an ongoing growth of tumor causing hypoxia and switched cell metabolism to glycolysis.

A mathematical model has been developed that takes into account all the changes described before. Modeling of the interaction of light with individual scatterers of different shapes and sizes was carried out using a Mie solution for spheres and cylinders. Tissue absorption coefficients for modeling were based on published data on the optical properties of blood, as well as information on the blood oxygenation and blood filling obtained during the in-vivo experiments. The Monte-Carlo simulation algorithm was developed to model the light propagation in media described by the mathematical models introduced before. A detailed description of this algorithm is provided in [2]. The comparison of simulation results with experimental data showed the correctness of the mathematical model of combined spectroscopic signal formation and diagnostic value of combined spectroscopic technique.

Acknowledgment: This work was supported by the Competitiveness Programm of NRNU MEPhI.

References

[1] Savelieva TA, Loshchenov VB, Goryainov SA, Shishkina LV, Potapov AA. A spectroscopic method for simultaneous determination of protoporphyrin IX and hemoglobin in the nerve tissues at intraoperative diagnosis. Russ J Gen Chem 2015;85(6):1549–57.

[2] Savelieva TA, Kalyagina NA, Kholodtsova MN, Loschenov VB, Goryainov SA, Potapov AA. Numerical modelling and in vivo analysis of fluorescent and laser light backscattered from glial brain tumours. Proc SPIE 2012;8230:82300L. doi: 10.1117/12.907444.

[4.02] Development of a fiber-optic scaffold for the glioblastoma diagnosis and prevention

Yuliya S. Maklygina¹, Aleksandr V. Borodkin¹, Gaukhar M. Yusubalieva² and Victor B. Loschenov^{1, 3}

¹A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

²V. P. Serbsky State Research Center of Forensic and Social Psychiatry, Ministry of Health, Kropotkinskij ln. 23, Moscow, 119991, Russian Federation

³National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Kashirskoe Highway 31, Moscow, 115409, Russian Federation

E-mail contact: us.samsonova@physics.msu.ru

Abstract: The main goal of the present research was the design of a unique fiber-optical multipurpose system created on the basis of porous optical fibers. These fiber-optical scaffolds were intended to build a structure, which can direct the promotion and settlement of glial cells growth. The system should also act as a port for the delivery of photosensitizers and laser radiation for the purpose of cellular process monitoring. As a result, the so developed system allows not only for implementing a standard method for fluorescence diagnostics but also for timely photodynamic therapy (PDT) of the probed area.

1 Introduction

Brain gliomas are known to invade and spread along the white matter channels and along the blood vessels [1]. Based on this observation, the present research aims to create ‘guide rails’ for tumors, consisting of porous optical fibers, which structurally imitate white matter channels and blood vessels. These guide rails are intended to direct the growth of localized tumor cells outwards, i.e. from the place of primary localization to a more easily accessible place outside the cerebral cortex, enabling the subsequent introduction of external effects targeted at the tumor cells destruction [1]. The monitoring of the cell growing process can be realized by fluorescence spectroscopy of such areas, with high fluorescence caused by an accumulation of a high molecule concentration of a photosensitizer in glioblastoma cells. This monitoring approach would give the observer a comprehensive understanding of the running processes within the probed area. In the case of gliomas, a directional growth of cancer cells from the intracranial area of the primary tumor to the outer part of the brain would be helpful to reduce the size of the primary tumor and to conduct therapeutic effect to the cancer cells, both timely and directed.

2 Materials and methods

In-vitro studies were performed on cell cultures of rat glioma C6 cells using confocal laser scanning microscopy. In-vivo studies were performed on experimental animals with induced malignant glioma in the intracranial region. During the study, scaffolds of different materials, shapes and sizes were developed and tested in experimental animals. The developed scaffolds were fixed subcutaneously on the skull of the experimental animals and served as a port for the local delivery of the photosensitizer and laser radiation. Evaluation of abscesses and rejection processes was made after implantation of the scaffolds using magnetic resonance imaging (MRI). The fluorescence monitoring of the tumor area was examined using a fiber spectrometer LESA-01-BIOSPEC [2] within the range of 400–1100 nm after fluorescence excitation at 632.8 nm and with a power density of ~100 mW/cm². PDT was realized using a wavelength of λ=675 nm, selected in accordance with the absorption spectra maxima of the phthalocyanine photosensitizers.

3 Results

In this study glioma C6 cell growth processes along the optical fibers were visualized using confocal laser scanning microscopy. These in-vitro studies showed that malignant glioma cells formed fast-growing agglomerates around the optical fibers and that they proliferated directionally along the fiber structures. The in-vivo studies showed that the malignant glioma cell growth from intracranial region to the extracranial part by means of the internal fiber optic structure of scaffolds reduced the intracranial tumor volume. During the study, scaffolds of different shapes and sizes were developed and tested in experimental animals. Successful testing could be confirmed by MRI (no abscesses and rejection of the scaffolds was observed) and allowed the determination of the optimal properties and the external dimensions of the brain scaffolds. The scaffolds developed were fixed subcutaneously on the skull of the experimental animals and served as a port for the local delivery of the photosensitizer and laser radiation for PDT, leading to malignant cell death. As a result of the PDT (phthalocyanine series photosensitizer, λ=675 nm) a highly therapeutic effect was observed and access to the tumor for monitoring processes could be ensured (Figure 1).

Figure 1:

MRI images of rat brain before therapy (left) and with scaffold after PDT at 675 nm (right).

4 Conclusion

The fiber-optic system developed allowed continuous monitoring of the processes taking place in the primary tumor showing a reduction in the volume of the primary tumor. The fiber-optic scaffold enabled a timely PDT. It is planned in the future to use infrared photosensitizers in order to achieve a higher therapeutic effect on the pathological processes of deep brain tissue localization. Such a combined approach may improve the treatment and prevent the spread of glioblastomas.

References

[1] Jain A, Betancur M, Patel GD, Valmikinathan CM, Mukhatyar VJ, Vakharia A, Pai SB, Brahma B, MacDonald TJ, Bellamkonda RV. Guiding intracortical brain tumour cells to an extracortical cytotoxic hydrogel using aligned polymeric nanofibres. Nat Mater 2014;13(3):308–16.

[2] Loschenov VB, Konov VI, Prokhorov AM. Photodynamic therapy and fluorescence diagnostics. Laser Phys 2000;10(6):1188–207.

[4.03] Spectral-temporal pulse construction for optimal nonlinear Raman brain imaging

E. A. Stepanov^{1, 2}, A. A. Lanin^{1, 2}, D. A. Sidorov-Biryukov^{1, 2}, A. B. Fedotov^{1, 2} and A. M. Zheltikov^{1, 2, 3, 4}

¹Physics Department, International Laser Center, M. V. Lomonosov Moscow State University, Moscow, 119992, Russian Federation

²Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region, 143025, Russian Federation

³Department of Physics and Astronomy, Texas A&M University, College Station, TX 77843, USA

⁴National Research Centre “Kurchatov Institute”, Akademika Kurchatova pl. 1, Moscow, 123182, Russian Federation

E-mail contact: ea.stepanov@physics.msu.ru

Abstract: In the present work, efficient strategies are proposed for pulse-width optimization that will be applicable for nonlinear Raman brain imaging. Ultrashort laser pulses with the spectral bandwidth, accurately matched against the bandwidth of molecular vibrations, are shown to provide a higher power of the total signal without reducing the sensitivity of tumor detection in brain tissues.

1 Pulse-width considerations for nonlinear Raman brain imaging

The past few years have witnessed a breakthrough in the development of stimulated Raman scattering (SRS) microscopy as an innovative approach for microscopy and bioimaging [1, 2]. In neuroscience, nonlinear Raman scattering offers an attractive alternative to fluorescent-protein-based approaches to brain imaging. While coherent anti-Stokes Raman scattering microscopy has been shown to hold much promise as a rapid, minimally invasive technique for molecular specific intraoperative optical diagnostics of brain lesions [3], SRS has been demonstrated to enable a fast label-free detection of brain tumors [4]. The Raman probing of molecular vibrations in some of the complex biological systems, such as the brain, may benefit from laser pulses with broader bandwidths, dictating the choice of femtosecond laser sources. With the spectral bandwidth of laser pulses accurately matched against the bandwidth of molecular vibrations, the coherent Raman signal is shown to be radically enhanced, enabling higher sensitivities and higher frame rates in nonlinear Raman brain imaging [5].

In the presented scheme of SRS microscopy, the Raman-induced loss is detected that the pump field experienced due to interaction with the Stokes field while passing through a sample. The relative SRS gain measured in this scheme, with the pump wave serving as a local oscillator, is given by:

where A_p and A_s are the amplitudes of the input pump and Stokes fields, respectively, and h(θ) is the Raman function. In Figure 1A, the SRS loss signal R is plotted, and calculated as a function of the driver pulse width τ≈τ_p≈τ_s, τ_p and τ_s being the pump and Stokes pulse widths, with the Raman function calculated in such a way as to match the Raman spectra of the white matter, cortex, and xenografts human brain tumor in a mouse brain [4].

Figure 1:

SRS loss signal plots. (A) The SRS loss signal at the frequency of the lipid peak, Ω_l≈2850 cm⁻¹, calculated as a function of the pulse width. (B) The ratio R_l/R_p of the lipid SRS loss signal to the protein SRS loss signal calculated as a function of the pulse width for the white matter (solid line), cortex (dashed line), and xenografts human brain tumor (dashed–dotted line) in a mouse brain.

The total SRS loss signal is seen to increase with the pulse bandwidth (Figure 1A), τ⁻¹, as long as τ⁻¹ remains smaller than the bandwidth Γ of a molecular vibration that dominates the Raman spectrum, saturating when τ⁻¹ becomes larger than Γ. However a high level of the total signal is insufficient for chemically specific imaging. The reliable detection of brain tumors requires not only a strong overall signal, but also a significant difference between the signals from a tumor and a normal brain tissue. Therefore, a method for the detection of this tumor by measuring the ratio R_l/R_p of the lipid SRS loss signal at Ω_l≈2850 cm⁻¹ to the protein SRS loss signal at Ω_p≈2930 cm⁻¹ and comparing the results of these measurements to the R_l/R_p ratio typical of a normal brain tissue is suggested. In Figure 1B, the ratio R_l/R_p calculated as a function of the pulse width for the white matter (solid line), cortex (dashed line), and xenografts human brain tumor (dashed−dotted line) in a mouse brain is presented. As long as the pulse width is longer than 500 fs, as our analysis shows, shorter pulses will give rise to a higher power of the total signal without the R_l/R_p ratio, quantifying the sensitivity of tumor detection in brain tissues.

2 Spectroscopic analysis of complex multicomponent systems

In the SRS imaging experiments presented here, a transform-limited pump and Stokes pulses with pulse widths of 150 and 200 fs were used. Such ultrashort pulses inevitably impose limitations on the spectral resolution in nonlinear optical microspectroscopy, making it difficult to resolve closely and overlapping lines in Raman spectra. However, the coherence of laser pulses suggests attractive solutions to this problem, allowing the spectral resolution of nonlinear microspectroscopy to be substantially improved by pulse chirping. A linear chirp is defined as a one-to-one map relating the frequency to the delay time τ between the laser pulses. With this map defined, the spectrum of a Raman active mode can be found by measuring the intensity of the nonlinear Raman signal as a function of τ [6].

Linearly chirped pump and Stokes pulses, used in the SRS spectroscopic experiments, had pulse widths of τ_p≈3.7 ps and τ_s≈3.2 ps. The chirp parameter was adjusted by varying the distance between the diffraction gratings in the stretchers, providing a spectral resolution of about 6 cm⁻¹, which is 17 times higher than the spectral resolution (100 cm⁻¹) attainable in the scheme with transform-limited pulses of the same pulse width. This offers a powerful tool for a reliable identification of molecular vibrations with close frequencies, enhancing the chemical selectivity of spectroscopic analysis of complex multicomponent systems [7].

Acknowledgments: This work was supported by the Russian State Targeted Program “Research and Development in Priority Areas of Development of the Russian Scientific and Technological Complex for 2014–2020” (Contract 14.607.21.0092 of November 21, 2014; unique identifier of applied research: RFMEFI60714X0092).

References

[1] Freudiger CW, Min W, Saar BG, Lu S, Holtom GR, He C, Tsai JC, Kang JX, Xie XS. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 2008;322(5909):1857–61.

[2] Ozeki Y, Umemura W, Otsuka Y, Satoh S, Hashimoto H, Sumimura K, Nishizawa N, Fukui K, Itoh K. High-speed molecular spectral imaging of tissue with stimulated Raman scattering. Nat Photon 2012;6:845–51.

[3] Evans CL, Xu X, Kesari S, Xie XS, Wong ST, Young GS. Chemically-selective imaging of brain structures with CARS microscopy. Opt Express 2007;15(19):12076–87.

[4] Ji M, Orringer DA, Freudiger CW, Ramkissoon S, Liu X, Lau D, Golby AJ, Norton I, Hayashi M, Agar NY, Young GS, Spino C, Santagata S, Camelo-Piragua S, Ligon KL, Sagher O, Xie XS. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci Transl Med 2013;5(201):201ra119.

[5] Lanin AA, Stepanov EA, Tikhonov RA, Sidorov-Biryukov DA, Fedotov AB, Zheltikov AM. Pulse-width considerations for nonlinear Raman brain imaging: whither the optimum? Laser Phys Lett 2015;12(11):115401.

[6] Lanin AA, Stepanov EA, Tikhonov RA, Sidorov-Biryukov DA, Fedotov AB, Zheltikov AM. Multimodal nonlinear Raman microspectroscopy with ultrashort chirped laser pulses. JETP Lett 2015;101:593–7.

[7] Lanin AA, Stepanov EA, Tikhonov RA, Sidorov-Biryukov DA, Fedotov AB, Zheltikov AM. A compact laser platform for nonlinear Raman microspectroscopy: multimodality through broad chirp tunability. J Raman Spectrosc 2016. doi: 10.1002/jrs.4860.

[4.04] Dual channel video fluorescence diagnostic system for intraoperational navigation during protoporphyrin IX photosensitized malignant tumor resection in central neural system

Maxim V. Loschenov¹, Alexander V. Borodkin¹, Denis A. Golbin², Sergey A. Goryaynov², Petr V. Zelenkov² and Alexander A. Potapov²

¹A. M. Prokhorov General Physics Institute of RAS, Vavilov Str. 38, Moscow, 119991, Russian Federation

²N. N. Burdenko Neurosurgery Institute, 16 4th Tverskaya-Yamskaya Street, Moscow, 125047, Russian Federation

E-mail contact:maxvl@mail.ru

Abstract: A novel neurosurgery fluorescence diagnostic system for navigation in photosensitized neural tissue, during surgery on malignant tumors has been developed. This system contains a beamsplitter adapter based on a dichroic mirror guiding the white light image to a high-sensitivity monochrome camera and the color image to a color camera. Both images are collected in a spectrally resolved manner, sent to a processor unit and displayed on a monitor. In the clinical situation, the presented system could indicate residual tumors including meningioma, neurofibroma, and glioblastoma.

1 Introduction

In the modern neurosurgery of malignant tumors, fluorescence navigation is one of the most sensitive and specific diagnostics methods to improve the efficacy of the surgical operation and therefore improve the survival time of the patient. Fluorescence diagnosis in the presence of selective photosensitizers can provide even better results in terms of sensitivity and specificity. Here a novel device is presented for the fluorescence navigation of photosensitized tumors together with its clinical application for different kinds of malignant tumors in the central nervous system. A fluorescence diagnostic system upgrade was developed for standard operation microscopes and endoscopes. The designed fluorescence system implements a number of innovative features, including fluorescence excitation at 635 nm and dual registration cameras.

2 Materials and methods

Patients were administered with the photosensitizer Alasens 3 h prior to the surgery. Transnasal operations were carried out using a Karl Storz endoscope; spinal tumors were operated by means of a Carl Zeiss OPMI microscope, and head and brain tumors were operated with a Carl Zeiss OPMI Pentero operation microscope. For the fluorescence diagnostics, a system was developed which consists of two light sources. The first was a light emitting diode-based white light (WL) source and the other a 635-nm laser diode-based source for fluorescence excitation. To detect the WL and fluorescence images, a dichroic mirror-based beam splitter with two digital cameras was developed: one color camera for registering the reflected WL image and one monochrome camera for the fluorescence signal. Both images were collected in real time, processed in a computer with software involving the use of the graphics processing unit (GPU) NVIDIA GeForce video card and displayed on the monitor in real time. To deliver the illumination light, a Y-shaped fiber-optic light guide was developed which collects light from both light sources mixing them together to illuminate the operating field.

3 Results

Experiments with meningioma that had infiltrated the nose cavity, neurofibroma in the spinal cord and glioblastoma in the head were carried out (Figure 1). As for the clinical result, strong fluorescence signal overlaying the video signal in natural colors for all three nosologies was obtained. As developed the system has the following innovative properties:

Figure 1:

Examples of method application in clinical conditions. 5-ALA was administered per os in the form of an aqueous solution at a dose of 20–30 mg/kg body weight 3 h prior to the procedure.

• Because of the fluorescence excitation in the red optical spectrum range, the tissue was scanned 3–5 times deeper than all the other devices available on the market. Furthermore, it was possible to perform fluorescence diagnostics in the presence of blood.

• The fluorescence navigation information could be superimposed on the real-time video signal from the tissue in natural colors.

• Using two cameras, the photosensitizer concentration could be assessed in the exposed tissue.

References

[1] Loshchenov M, Zelenkov P, Potapov A, Goryajnov S, Borodkin A. Endoscopic fluorescence visualization of 5-ALA photosensitized central nervous system tumors in the neural tissue transparency spectral range. Photonics Lasers Med 2014;3(2):159–70.

[2] Markwardt NA, Haj-Hosseini N, Hollnburger B, Stepp H, Zelenkov P, Rühm A. 405 nm versus 633 nm for protoporphyrin IX excitation in fluorescence-guided stereotactic biopsy of brain tumors. J Biophotonics 2016;9(9):901–12.

Author Index

[1.01] – [4.04] represents the abstracts numbers

Abdulvapova, ZN. [2.12]

Akhlyustina, EV. [1.10], [1.11]

Amouroux, M. [2.11]

Ardamakova, A. [2.04]

Astafiev, AA. [2.01]

Astaf’eva, LG. [1.02]

Balla, VK. [1.01]

Barun, VV. [2.06], [2.08]

Blondel, WCPM. [2.11]

Bolshunov, A. [2.04]

Bondarenko, ON. [2.12]

Borodkin, AV. [1.10], [2.05], [4.02], [4.04]

Boruleva, EA. [1.05]

Bunkin, AF. [1.08]

Bystrov, FG. [1.09]

Cherkasova, OP. [2.10]

Chuhonsky, AI. [1.07]

Daul, C. [2.11]

Dolgushin, BI. [3.02]

Dolgushin, MB. [3.02]

Dzhagarov, BM. [1.04]

Farrakhova, DS. [1.03], [1.11]

Fedotov, AB. [4.03]

Filonenko, EV. [1.10], [3.03]

Galstyan, GR. [2.12]

Ghazaryan, RK. [1.04]

Golbin, DA. [4.04]

Goryaynov, AA. [4.01], [4.04]

Grachev, PV. [2.05], [2.12]

Gräfe, S. [3.01]

Grigoreva, EYu. [3.02]

Grishin, MYa. [1.08]

Gyulkhandanyan, AG. [1.04]

Gyulkhandanyan, GV. [1.04]

Ivannikov, SV. [2.07]

Ivanov, AP. [2.06], [2.08]

Kaprin, AD. [3.03]

Karpova, OV. [1.08]

Karu, TI. [2.03]

Kholodtsova, MN. [1.06], [2.11]

Korobtsov, AV. [2.02]

Kotova, SP. [2.02]

Kruchenok, YuV. [1.07]

Kudryavtseva, AD. [1.08]

Kundu, B. [1.01]

Kuneva, AA. [1.10]

Kurlov, VN. [3.04]

Kuznetsova, JO. [1.03]

Lanin, AA. [4.03]

Larichev, A. [2.04]

Lednev, VN. [1.08]

Lepeshkevich, SV. [1.04]

Linkov, KG. [2.09]

Loschenov, MV. [3.03], [4.04]

Loschenov, VB. [1.01], [1.06], [1.09], [2.07], [2.11], [3.01], [3.02], [4.02]

Losevsky, NN. [2.02]

Lytkin, A. [2.04]

Makarov, VI. [1.06], [1.09], [1.11], [2.07]

Maklygina, YuS. [1.01], [1.10], [3.02], [4.02]

Manteifel, VM. [2.03]

Mayorova, AM. [2.02]

Meerovich, GA. [1.10]

Mironova, TV. [1.08]

Mukhin, AE. [2.05]

Nadtochenko, VA. [2.01]

Nazarov, MM. [2.10]

Nemkovich, NA. [1.07]

Nevzorov, DI. [3.02]

Osychenko, AA. [2.01]

Parkhats, MV. [1.04]

Pershin, SM. [1.08]

Petrova, EK. [1.08]

Pominova, DV. [1.06], [1.11]

Potapov, AA. [4.01], [4.04]

Pyatibrat, LV. [2.03]

Rakotomanga, P. [2.11]

Romanishkin, ID. [2.07]

Ryabova, AV. [1.06], [1.10], [1.11], [2.07], [3.01], [3.02]

Rybakova, PA. [1.10]

Samagin, SA. [2.02]

Sargsyan, HH. [1.04]

Savelieva, TA. [4.01]

Savitsky, AP. [1.05]

Scalfi-Happ, C. [3.01]

Semenova, LA. [2.07]

Shakhov, AM. [2.01]

Shanko, YuG. [1.07]

Sharova, AS. [1.01]

Shikunova, IA. [2.09], [3.04]

Shkurinov, AP. [2.10]

Shmeleva, S. [2.04]

Sidorov-Biryukov, DA. [4.03]

Simonova, V. [2.04]

Sipliviy, V. [2.04]

Smorchkov, MM. [2.07]

Sobchuk, AN. [1.07]

Sokolov, EN. [3.02]

Soussen, C. [2.11]

Stasheuski, AS. [1.04]

Steiner, R. [1.01], [3.01]

Stepanov, EA. [4.03]

Stranadko, EF. [2.05], [2.07]

Strokov, MA. [1.08]

Tcherniega, NV. [1.08]

Titov, AA. [2.01]

Tonenkov, AM. [2.07]

Urlova, AN. [3.03]

Volkov, VV. [2.09], [3.04]

Wittig, R. [3.01]

Yakovlev, DV. [1.10]

Yassin, MG. [1.03]

Yusubalieva, GM. [4.02]

Zalesskaya, GA. [1.02]

Zalessky, AD. [2.01]

Zelenkov, PV. [4.04]

Zemskov, KI. [1.08]

Zharova, TA. [2.07]

Zheltikov, AM. [4.03]

Zherdeva, VV. [1.05]