Volume 63, Issue 5 - Special Issue: Optical Imaging Methods in Medicine / Guest Editors: Werner Nahm, Christoph Hornberger, Ute Morgenstern and Stephan B. Sobottka

Optical imaging of biological tissue in vivo at multiple wavelengths in the near-infrared (NIR) spectral range can be achieved with picosecond time resolution at high sensitivity by time-correlated single photon counting. Measuring and analyzing the distribution of times of flight of photons randomly propagated through the tissue has been applied for diffuse optical imaging and spectroscopy, e.g. of human breast tissue and of the brain. In this article, we review the main features and the potential of NIR multispectral imaging with picosecond time resolution and illustrate them by exemplar applications in these fields. In particular, we discuss the experimental methods developed at the Physikalisch-Technische Bundesanstalt (PTB) to record optical mammograms and to quantify the absorption and scattering properties from which hemoglobin concentration and oxygen saturation of healthy and diseased breast tissue have been derived by combining picosecond time-domain and spectral information. Furthermore, optical images of functional brain activation were obtained by a non-contact scanning device exploiting the null source-detector separation approach which takes advantage of the picosecond time resolution as well. The recorded time traces of changes in the oxy- and deoxyhemoglobin concentrations during a motor stimulation investigation show a localized response from the brain.

Worldwide, chronic wounds are still a major and increasing problem area in medicine with protracted suffering of patients and enormous costs. Beside conventional wound treatment, for instance kinds of oxygen therapy and cold plasma technology have been tested, providing an improvement in the perfusion of wounds and their healing potential, but these methods are unfortunately not sufficiently validated and accepted for clinical practice to date. Using hyperspectral imaging technology in the visible (VIS) and near infrared (NIR) region with high spectral and spatial resolution, perfusion parameters of tissue and wounds can be determined. We present a new compact hyperspectral camera which can be used in clinical practice. From hyperspectral data the hemoglobin oxygenation (StO 2 ), the relative concentration of hemoglobin [tissue hemoglobin index (THI)] and the so-called NIR-perfusion index can be determined. The first two parameters are calculated from the VIS-part of the spectrum and represent the perfusion of superficial tissue layers, whereas the NIR-perfusion index is calculated from the NIR-part representing the perfusion in deeper layers. First clinical measurements of transplanted flaps and chronic ulcer wounds show, that the perfusion level can be determined quantitatively allowing sensitive evaluation and monitoring for an optimization of the wound treatment planning and for validation of new treatment methods.

Hyperspectral imaging (HSI) has become a sophisticated technique in modern applications such as food analyses, recycling technology, medicine, pharmacy and forensic science. It allows one to analyse both spatial and spectral information from an object. But hyperspectral cameras are still expensive due to their extended wavelength range. The development of new light-emitting diodes (LED) in the recent past enables another approach to HSI using a monochrome camera in combination with a LED-based illumination. However, such a system has a lower spectral resolution. Additionally, the growing supply of LED on the market complicates the selection of LED. In this paper, we propose a new time efficient selection method for the design process of an illumination. It chooses an optimised LED combination from an existing database to match a predefined spectral power distribution. Therefore, an algorithm is used to evaluate various LED combinations. Furthermore, the method considers the spectral behaviour of each LED in dependence of forward current and temperature of the solder point. Our method has already shown promise during the selection process for even spectral distributions which is demonstrated in the study. Additionally, we will show its potential for HSI illuminations.

Pulse oximetry for arterial oxygenation monitoring and tissue oximetry for monitoring of cerebral oxygenation or muscle oxygenation are based on quantitative in vivo diffuse optical spectroscopy. However, in both cases the information on absolute or relative concentration of human tissue constituents and especially on hemoglobin oxygenation can often not be retrieved by model-based analysis. An in vivo calibration against an accepted reference measurement can be a practical alternative. Pulse oximeters and most of commercial cerebral tissue oximeters rely on empirical calibration based on invasive controlled human desaturation studies. As invasive in vivo tests on healthy subjects are ethically disputable and should be limited to exceptional cases this calibration practice is unsatisfactory. We present the current status and problems of calibration and validation in pulse oximetry and cerebral tissue oximetry including the pros and cons of in vivo as well as in vitro methods. We emphasize various digital and physical phantom approaches and discuss the prospects of their application and possible further developments.

Blood perfusion is the supply of tissue with blood, and oxygen is a key factor in the field of minor and major wound healing. Reduced perfusion of a wound bed or transplant often causes various complications. Reliable methods for an objective evaluation of perfusion status are still lacking, and insufficient perfusion may remain undiscovered, resulting in chronic processes and failing transplants. Hyperspectral imaging (HSI) represents a novel method with increasing importance for clinical practice. Therefore, methods, software and algorithms for a new HSI system are presented which can be used to observe tissue oxygenation and other parameters that are of importance in supervising healing processes. This could offer an improved insight into wound perfusion allowing timely intervention.

The lack of donor grafts is a severe problem in transplantation medicine. Hence, the improved preservation of existing and the usage of organs that were deemed untransplantable is as urgent as ever. The development of novel preservation techniques has come into focus. A promising alternative to traditional cold storage is normothermic machine perfusion (NMP), which provides the benefit of improving the organs’ viability and of assessing the organs’ status under physiological conditions. For this purpose, methods for evaluating organ parameters have yet to be developed. In a previous study, we determined the tissue oxygen saturation (StO 2 ) of kidneys during NMP with hyperspectral imaging (HSI) based on a discrete wavelength (DW) algorithm. The aim of the current study was to identify a more accurate algorithm for StO 2 calculation. A literature search revealed three candidates to test: a DW algorithm and two full spectral algorithms – area under a curve and partial least square regression (PLSR). After obtaining suitable calibration data to train each algorithm, they were evaluated during NMP. The wavelength range from 590 to 800 nm was found to be appropriate for analyzing StO 2 of kidneys during NMP. The PLSR method shows good results in analyzing the tissues’ oxygen status in perfusion experiments.

The resection of brain tumor requires a precise distinction between eloquent areas of the brain and pathological tumor tissue in order to improve the extent of resection as well as the patient’s progression free survival time. In this study, we discuss mathematical tools necessary to recognize neural activity using thermal imaging cameras. The main contribution to thermal radiation of the exposed human cortex is regional cerebral blood flow (CBF). In fact, neurovascular coupling links neural activity to changes in regional CBF which in turn affects the cortical temperature. We propose a statistically sound framework to visualize neural activity of the primary somatosensory cortex. The framework incorporates a priori known experimental conditions such as the thermal response to neural activity as well as unrelated effects induced by random neural activity and autoregulation. These experimental conditions can be adopted to certain electrical stimulation protocols so that the framework allows to unveil arbitrary evoked neural activity. The method was applied to semisynthetic as well as two intraoperative cases with promising results as we were able to map the eloquent sensory cortex with high sensitivity. Furthermore, the results were validated by anatomical localization and electrophysiological measurements.

The intraoperative identification of normal and anomalous brain tissue can be disturbed by pulsatile brain motion and movements of the patient and surgery devices. The performance of four motion correction methods are compared in this paper: Two intensity-based, applying optical flow algorithms, and two feature-based, which take corner features into account to track brain motion. The target registration error with manually selected marking points and the temporal standard deviation of intensity were analyzed in the evaluation. The results reveal the potential of the two types of methods.

Label-free multiphoton imaging constitutes a promising technique for clinical diagnosis and therapeutic monitoring. Corpora amylacea (CoA) are starch-like structures often found in the diseased brain, whose origin and role in nervous pathologies are still a matter of debate. Recently, CoA in the diseased human hippocampus were found to be second harmonic generation (SHG) active. Here, we show that CoA formed in other parts of the diseased brain and in brain neoplasms display a similar SHG activity. The SHG pattern of CoA depended on laser polarization, indicating that a radial structure is responsible for their nonlinear activity. Vibrational spectroscopy was used to study the biochemistry underlying the SHG activity. Infrared (IR) and Raman spectroscopy showed that CoA contain polyglucosans that are biochemically similar to glycogen, but with an unusual structure that is similar to amylopectin, which justifies the nonlinear activity of CoA. Our findings explain the SHG activity of CoA and demonstrate that CoA in the pathological brain are amenable to label-free multiphoton imaging. Further research will clarify whether intraoperative assessment of CoA can be diagnostically exploited.

Brain tumor resection is even today one of the most challenging disciplines in neurosurgery. The current state of the art for the identification of tumor tissue during the surgical procedure comprises a wide variety of different tools, each with its own limitations and drawbacks. In this paper, we present a novel approach, the use of optical imaging in connection with direct electrical cortical stimulation (DCS), for identification of impaired tumor tissue and functional intact normal brain tissue under intraoperative conditions. Measurements with an optical imaging setup were performed as a proof of concept on three patients who underwent tumor resection of superficial gliomas. Direct electrical stimulations were applied on tumor tissue and surrounding brain tissue in each patient and characteristic features from the observed changes in the optical properties were compared between the different groups. The results reveal that in all patients a differentiation between non-functional tumor tissue and functional intact brain tissue was possible, and the technique might be a useful clinical tool in the future.

The detection of the beginning demineralization process of dental hard tissue remains a challenging task in dentistry. As an alternative to bitewing radiographs, optical and spectroscopic technologies showed promising results for caries diagnosis. The aim of the present work is to give an overview of optical and spectroscopic properties of healthy and carious human teeth in vitro by means of Raman spectroscopy (RS), optical coherence tomography (OCT) and hyperspectral imaging (HSI). OCT was able to represent microstructural changes below the enamel surface and revealed increased scattering for white spot lesions and a white scattering trail for deeper lesions. HSI showed similar absorbance characteristics for healthy and demineralized enamel over the entire spectrum and a characteristic absorbance peak at 550 nm for discolored lesions. Already at early carious stages (white spot), we found a distinct loss of hydroxylapatite-related intensity at 959 cm −1 in demineralized regions with RS. Healthy and demineralized tooth surfaces can be distinguished at different signal levels by means of RS, OCT and HSI. The presented modalities provide additional information to the current clinical diagnosis of caries such as microstructural changes, quantification of the demineralization and imaging of caries-related chemical changes.

An important clinical potential of cold atmospheric plasma (CAP) lies in tumor and wound treatment, whereby the last-mentioned is well-referenced already. However, the underlying mechanisms of improved wound healing have not been sufficiently clarified yet, in particular the influence of CAP on microcirculation. Hyperspectral imaging (HSI) enables the visualization of microcirculation of large tissue areas, thus this technique seems to be a candidate to examine CAP effects on perfusion and oxygen saturation in wounds. During clinical wound management, one chronic wound caused by peripheral arterial occlusive disease and one acute wound after surgical removal of cervical lymph nodes were examined using HSI before and after CAP treatment. HSI was able to demonstrate CAP effects on microcirculation showing a relevant increase of superficial and deeper cutaneous oxygen saturation together with elevated hemoglobin concentration in treated and also surrounding wound area. For the first time, it was shown that CAP improves the superficial and deeper oxygenation and hemoglobin perfusion in and around the treated area of acute and chronic wounds. This effect may contribute to healing support by CAP in wounds. HSI seems suitable for evaluating and monitoring CAP effects in clinical settings.

There is a lack of imaging tools for the evaluation of spatial alterations in microcirculation including blood oxygen saturation and hemoglobin distribution but recent innovative developments in hyperspectral technology may offer a solution. We examined different hemodynamic disorders in patients suffering from scleroderma, Dupuytren surgery, chronic foot ulcera and skin infections. Superficial and deeper blood oxygen saturation, hemoglobin distribution and water content were determined using hyperspectral imaging (HSI). In the patient with scleroderma, distinct cutaneous low perfused regions correlated with macroscopic skin aspects and seem to be potential therapy control marker. With HSI accurate clinical evaluation of a macroscopic conspicuous wound after Dupuytren surgery was possible and influenced further surveillance decisions. HSI clearly revealed the spatial geometry and also the clinically related perfusion parameters of abscess formation and chronic ulcer wounds. The hemodynamically relevant parameters like blood oxygen saturation (1 mm to approx. 6 mm subcutaneous), total hemoglobin distribution and tissue water content can be easily determined and visualized with HSI in near real time. Hence, this technique seems to be suitable for routine diagnostics of acute and chronic wounds as well as for the examination of systemic hemodynamic disturbances. Special indications may be transplant surveillance and monitoring of therapeutical interventions.

Over the last few years, the contactless acquisition of cardiovascular parameters using cameras has gained immense attention. The technique provides an optical means to acquire cardiovascular information in a very convenient way. This review provides an overview on the technique’s background and current realizations. Besides giving detailed information on the most widespread application of the technique, namely the contactless acquisition of heart rate, we outline further concepts and we critically discuss the current state.