Enhancing the generating and collecting efficiency of single particle upconverting luminescence at low power excitation

1 Introduction

Upconversion nanoparticles (UCNPs) possess the ability to convert low-energy infrared (IR) photons into high-energy visible or ultraviolet (UV) photons. This unique anti-Stokes feature has enabled various applications over the past decade, spanning from solar energy harvesting, superresolution microscopy, and anticounterfeit luminescent imprinting to agricultural crop care [1], [2], [3], [4], [5], [6]. Among the various nanoparticle systems developed thus far, semiconductor nanocrystals, 2D materials, and quantum dots (QDs) are widely used [7], [8], [9], [10]. UCNPs typically consist of a NaYF₄ crystalline host matrix doped with rare-earth-based lanthanide sensitizer and activator. Such particles can be designed and synthesized based on desirable physicochemical characteristics such as sizes, shapes, optical properties, and magnetic properties [11], [12], [13], [14].

The development of near-IR (NIR)-responsive photonic materials is of great scientific interest, as life science, materials science, and aerospace industries can all take advantage of the penetrative ability of NIR light for invisible, high-precision remote activation of materials [1]. UCNPs can efficiently convert NIR light to UV or visible light and have attracted recent attention in biomedical applications for this ability. Nanoparticles feature efficient cellular uptake while retaining low cytotoxicity and display high optical penetrating power in deep tissue with minimal background noise [15], [16]. In addition, UCNPs exhibit neither photoblinking on millisecond and second-time scales nor photobleaching even with hours of continuous excitation. As a result, UCNPs are becoming one of the most promising nanoparticle systems for in vivo biological imaging, biosensing, and nanomedicine, with continuing efforts to improve their properties including the design of new synthetic strategies [17].

Single nanoparticle imaging is essential for cell tracking. Studies include long-term tracking of UCNPs in live cells [18], deep subdiffraction fluorescence microscopy [19], single-molecule tracking under lower irradiance [20], using agarose-gel electrophoresis to make background-free labels [21], and improving upconverting fluorescence with natural biomicrolens [22]. Single nanoparticle imaging is essential for the detection of single-cell imaging in techniques involving cellular tracking and providing accurate cellular movement.

For bioimaging applications in vitro, high excitation power is not recommended. Imaging and longitudinal real-time tracking of live cells prefer low power excitation. For example, power-dependent analysis is often used in various studies, including investigating energy transference relations between dopants [23], economically feasible UCNPs in anticounterfeiting ink printing [24], thin-film synthesis using electrodeposition [25], and upconverting luminescence depletion phenomena via simultaneous wavelength excitation [26]. Therefore, in practical usage of UCNPs in bioinformatics, maximizing luminescence efficiencies is of great significance. In this article, emphasis is placed on such augmentation of upconversion intensity in response to power irradiance. In this study, Tm³⁺ ions are chosen as dopants due to their emission wavelength of ~800 nm, which is within the “NIR biological window”. This allows for low autofluorescence and reduced photon-damage effects but also offers deep light penetration and low light scattering [27], [28].

Many studies have aimed to improve upconverting luminescence by the advancement of synthesis methodology and instrumental characterization at nanoscale over the last decades. Enhancement strategies include optimizing dopant concentrations and crystal hosts, coating inert/active shells around UCNPs, tuning the sizes of UCNPs, and coupling UCNPs with plasmonic noble metal nanostructures. One of the most commonly used of these methods is the passivation of the particle’s surface with an inert shell such as NaYF₄, NaGdF₄, NaLuF₄, or CaF₂ to form a dopant free layer. This can block the pathway of energy migration to surface quenchers [29], [30] (Figure S3). In the synthesis of UCNPs, a higher dopant concentration of sensitizer ions will enhance luminescence. However, an oversaturation of sensitizer concentration will decrease luminescence [31]. This concentration quenching consequence is an important factor to take into consideration when addressing dopant concentrations in such particles [32], [33]. The addition of an inactive shell surrounding the particle provides a protective layer that prevents luminescence quenching associated with surface defects on the core particles but at a cost of increasing nanoparticle size [34]. It is found that typical lanthanide-based UCNPs containing smaller particles below 10 nm in size endure more difficulty in displaying efficient upconverting luminescence due to their cubic crystal lattice structure [35]. However, nanoparticle size for efficient cellular uptake favors smaller sizes, which can decrease the toxicity of the particles within subcellular regions and increase biocompatibility [36], [37].

There are several reports on acquiring high-contrast cellular imaging through testing with a customized confocal microscope with a moderate resolution in response to the saturation effect of upconverting luminescence [38]. The saturation effect is easily achieved with high numerical aperture (NA) confocal microscopy with a power density in the range from 10 kW/cm² to 1 MW/cm² [39]. For live cell imaging, a probe with high quantum efficiency and low saturated excitation threshold power will benefit from long-term live cell imaging or single-particle tracking.

In this article, multifarious methods to improve luminescence intensity in UCNPs are investigated. More power efficient UCNPs are synthesized by scrutinizing dopant concentration, particle size, and core-shell influence. The variation of luminescence efficiency with respect to each of these attributes is also quantified and compared. Emphasis is given to the parameter values at which higher-performing nanoparticles undergo increased luminescence at lower power density than their weaker-performing counterparts. We explored UCNPs with highly doped Yb³⁺ sensitizer ions and Tm³⁺ activator dopants in a NaYF₄ host and their core-shell structures. This combination of dopants is typical lanthanide dopant used in UCNP research and has been used in bioinformatic investigations [40], [41]. With a dopant concentration of 60% Yb³⁺, 8% Tm³⁺ in a NaYF₄ host, we present about seven times more efficient UCNP that will be well suited for applications in live cell imaging.

2 Results and discussion

There are various methods to increase upconverting luminescence with concentrated samples. We also found multiple methods to increase upconverting luminescence for a single nanoparticle [33], [42], as shown in Figure 1. Nanoparticle size is an important aspect that can influence upconverting luminescence, as different nanoparticle sizes adjust the surface/volume ratio and directly influence the defects/unit. The size of nanoparticles is controlled below 50 nm in consideration toward use for bioapplications. We use optimized synthesis methods to synthesize individual nanoparticles within similar sizes, as shown in Figures S1 and S2. Similar sizes will maintain the same surface/volume ratio, which will greatly eliminate the influence of surface defects.

Figure 1:

Multiple methods to increase upconverting luminescence for a single nanoparticle.

(A) Schematic illustration of the system setup for customized LSM. The excitation source is a 980 nm CW laser (power density of 30 MW/cm²) that is focused on the sample through an oil immersion objective lens (100×, NA 1.4, Olympus). The dichroic mirror into the detector reflects the emission from the sample. A large field PMT (Multialkali Amplified PMT, PMT1001, Thorlabs) detector is employed to collect emission fluorescence photons. Scanning is achieved by two conjugated placed galvanometer mirrors. A motorized wave plate and a polarizing beam splitter (PBS) are employed to control the power, and a cover glass and photon diode (PD) is used to measure the power on the sample after objective remotely. (B) Spatial confinement of generated fluorescence generation with upconverting nonlinear excitation. In one photon fluorescence process, the whole illumination cone will generate a fluorescence signal, but the upconverting nonlinear generated signal only localized the focal spot. Upconverting nonlinear process generated a fluorescence photon by absorbing more than two photons, whereas one photon fluorescence process case absorbs one photon. TEM images and microscopy quantitative measurement of the whole upconverting spectrum luminescence emission of single UCNPs of (C) NaYF₄: 4% Tm³⁺, 20% Yb³⁺ UCNPs; (D) NaYF₄: 4% Tm³⁺, 45% Yb³⁺ UCNPs; (E) NaYF₄: 8% Tm³⁺, 20% Yb³⁺ UCNPs; (F) NaYF₄: 4% Tm³⁺, 20% Yb³⁺ @ NaYF₄ UCNPs; and (G) NaYF₄: 8% Tm³⁺, 60% Yb³⁺ @ NaYF₄ UCNPs. Scale bar in TEM and fluorescence images, 100 nm and 1 μm, respectively. The pixel dwell time in fluorescence image acquisition is 200 μs. Comparison of integrated single-particle upconverting luminescence emissions of (H) core (4% Tm³⁺, 20% Yb³⁺) and core-shell and (i) NaYF₄: 4% Tm³⁺, 20% Yb³⁺ vs. NaYF₄: 8% Tm³⁺, 60% Yb³⁺ @ NaYF₄ UCNPs under different excitation power.

As Yb³⁺ acts as a sensitizer that can transfer 980 nm phonon energy to rare-earth ions, it is assumed that high sensitizer and rare-earth ion dopant concentrations will make upconverting luminescence much brighter under high excitation power [2]. As shown in Figure 1D, 45% Yb³⁺-doped UCNP is 1.33 times higher than 20% Yb³⁺ (Figure 1C)-doped particles under 1 MW/cm² power density. Increasing Yb³⁺ concentration will decrease the atomic distance from sensitizers to activators; thus, energy transfer will be more efficient when high excitation power continually transfers energy from Yb³⁺ to activators. The luminescence intensity of single nanoparticles with different dopant concentrations has been measured using a customized laser scanning microscope (LSM; Figure 1A). The luminescence intensity of a single nanoparticle is represented as the maximum pixel value for each Gaussian spot.

Based on a previous report, a brighter upconverting luminescence can be obtained under high excitation power in high activator concentration (4% Tm³⁺) codoped with 20% Yb³⁺ UCNPs [39]. We tried to use a higher activator concentration to further increase luminescence, which is 8% Tm³⁺-doped UCNPs. As shown in Figure 1E, the same trend occurs such that luminescence is enhanced 1.64 times higher.

Concentration quenching also can be overcome at high excitation powers by introducing a thin layer of an inert shell onto the surface of the nanoparticle. The high-resolution transmission electron microscopy (HRTEM) core and core-shell characterization in Figure S4 and the inductively coupled plasma-optical emission spectrometry composition data for core-shell versus core-only nanoparticles are shown in Table S2 to confirm the synthesis of this structure. In Figure 1F, the core-shell particle increases luminescence by 1.48 times compared to the particles, which only contain the core structure. Yb³⁺-Yb³⁺ energy migration to surface defects, which lead to quenching, is primarily responsible for the depopulation of core UCNPs. This result demonstrates that, at single-particle laser powers, quenching typically associated with high Yb³⁺ content can be suppressed by external inert shells.

At the same time, Tm³⁺ ions mainly produce ³H₄→³H₆ transitions at low excitation power, but a different situation appears when excited under high power. The transitions shown in Figure 1H, blue wavelength (¹D₂→³F₄, ¹G₄→→ ³H₆), red wavelength (¹G₄→→ ³F₄), and NIR (¹D₂→³F₂, ¹D₂→→ ³F₃, ³H₄ →³H₆), are all verified in Figure 1B. From the different wavelength emission changes under different excitation power, we find that the electron population on ¹D₂ and ¹G₄ states increased faster than ³H₄ state with increasing excitation power. The ³H₄ state releases the 800 nm emission; however, when additional emission energy is transferred from Yb³⁺ under high excitation power, ³H₄ state eventually achieves full capacity and is excited to higher ¹D₂ and ¹G₄ states.

It is worth noting that the luminescence intensity ratio difference between core-shell and core-only UCNPs is higher under 10 kW/cm² (3.65 times) than 1 MW/cm² (1.48 times), as shown in Figure 1H. Low power density is required for in vivo imaging; therefore, importance is placed on attaining the same luminescence intensity with lower excitation power density. Based on all listed luminescence enhancement methods, such intensity with low power density is produced using higher sensitizer and activator concentration combinations along with a core-shell structure. As Figure 1I shows, the luminescence of the core-shell structure with high dopant concentrations (8% Tm³⁺, 60% Yb³⁺, as shown in Figure 1G) is 6.83 times higher than normal UCNP particles under 0.05 MW/cm².

To systematically compare the brightness of all the improved methods, we measured the power-dependent luminescence curves for single particles from 5 kW/cm² to 10 MW/cm², as shown in Figure 2C, D, G, and H. The quantitative intensity images are shown in Figure 2A, B, E, and F and every image is taken within the same area for each sample. From previous recordings of data obtained from the sensitizer concentration and power dependence testing, it is foreseen that luminescence would be enhanced with the increase in excitation power. Figure 2A and D (4% Tm³⁺-doped UCNPs) shows that higher sensitizer concentrations will not only increase luminescence intensity under each excitation power but can also shift power dependence curve to lower power. In other words, we can use lower power excitation with higher sensitizer concentration UCNPs to get the same intensity as that using high power excitation with lower sensitizer concentration. Figure 2B and F (8% Tm³⁺-doped UCNPs) shows the same trends as 4% Tm³⁺ particles. However, higher Tm³⁺ dopant concentrations will increase luminescence intensity under each excitation power.

Figure 2:

Microscopy quantitative measurement of the whole upconverting spectrum luminescence emission.

(A) single 4% Tm³⁺ UCNPs vs. Yb³⁺ concentration, (B) single 8% Tm³⁺ UCNPs vs. Yb³⁺ concentration, (E) single 4% Tm³⁺ core-shell UCNPs vs. Yb³⁺ concentration, and (F) single 8% Tm³⁺ core-shell UCNPs vs. Yb³⁺ concentration. The power-dependent saturation curves of (C) single 4% Tm³⁺ UCNPs vs. Yb³⁺ concentration, (D) single 8% Tm³⁺ UCNPs vs. Yb³⁺ concentration, (G) single 4% Tm³⁺ core-shell UCNPs vs. Yb³⁺ concentration, and (H) single 8% Tm³⁺ core-shell UCNPs vs. Yb³⁺ concentration. The saturation curves under different power densities from 5 kW/cm² to 10 MW/cm² obtained with large field PMT detector-based LSM. A log scale is used for the y-axis in the left panels of (C and D) and (G and H), and a linear y-scale is employed in the right panels of (C and D) and (G and H). Scale bar, 1 μm; pixel dwell time, 200 μs. Error bars represent standard deviation from the mean.

Another remarkable phenomenon is the power dependence slope increase by introducing a core-shell structure, in which surface defects are decreased. We can use 0.1 MW/cm² for 4% Tm³⁺/45% Yb³⁺ core-shell particles to obtain the same highest luminescence intensity of the related core particle under 0.01 MW/cm². Comparing 4% Tm³⁺ UCNPs to 8% Tm³⁺-doped particles, the power dependence slope for 8% Tm³⁺ is higher than 4% Tm³⁺ UCNPs. This indicates that 8% Tm³⁺-doped UCNP can reach its saturation point at a much lower power than 4% Tm³⁺ UCNP. Also, 8% Tm³⁺-doped UCNP is able to reach the same luminescence intensity using much lower excitation power. For instance, the highest luminescence intensity of NaYF₄: 4% Tm³⁺/20% Yb³⁺ @ NaYF₄ UCNP is under 1 MW/cm², and the same intensity can be reached under 0.02 MW/cm² by NaYF₄: 8% Tm³⁺/60% Yb³⁺ @ NaYF₄ UCNP. Based on these results, excitation power can be decreased by more than one order of magnitude using high Tm³⁺ with high Yb³⁺ dopants. It is indicative that short distances between sensitizer and activator ions as well as high power excitations that increase the electron populations of excited states play an important role in protecting the surface quenching of same-sized UCNPs.

Spatial resolution and collection efficiency are great advantages of large field photomultiplier tube (PMT) detector-based LSM for high nonlinear upconverting fluorescence nanoparticles. Several groups have reported their results of single upconverting nanoparticle characterization employing confocal microscopy [20], [43], [44], [45], [46] as well as confocal based laser scanning subdiffraction microscopy [2], [19], [47]. The merits of nonlinear contrast mechanisms are that the signal (S) depends supralinearly (SμIⁿ) to the excitation light intensity (I). As a result, when focusing on the laser beam through a microscope objective, high-order nonlinearity absorption is spatially confined to the perifocal region. The high-order nonlinear property of upconverting luminescence shown in Figure 2 can benefit not only the spatial resolution of LSM but also the out-of-focus background, which could be suppressed by high-order nonlinear generated fluorescence. Thus, a pinhole is not needed to reject out-of-focus background. Of equal importance to the maximization of signal generation is the optimization of collection and detection efficiencies. The pinhole will also degrade the collection efficiency of the generated fluorescence once it is not well coupled. High-order nonlinear microscopes (Figure 1A) use PMTs, available with large sensitive areas and reasonable quantum efficiencies rather than the pinhole-based detector and come with a high spatial resolution of nonlinear upconverting fluorescence nanoparticles.

The resolution of confocal images degrades as laser power increases because excitation power has exceeded the nonlinear threshold. For microscopy, the image of any object is acquired by convolving the point spread function (PSF) of the optical system [48]. For confocal microscopy, PSF_confocal=PSF_illumination×PSF_detection. For Nth order nonlinearity LSM with whole area detection, the effective PSF, PSF_N-order=(PSF_illumination)^N=PSF^N (u, v) [48]. As shown in Figure 2, log-scale power dependence fluorescence saturated curves have a higher slope under low power density. For example, 4% Tm³⁺, 20% Yb³⁺ core UCNPs have a slope of ~1.6 below 1 MW/cm² (saturated intensity), and 8% Tm³⁺, 60% Yb³⁺ core-shell UCNPs have a slope of ~2 with below the saturated intensity. In Figure 3, the image under lower power density of 5 kW/cm² has a much smaller full-width at half-maximum (FWHM) of 279.0 nm compared to the case with 100 kW/cm² (saturated absorbed). Note that 4% Tm³⁺, 20% Yb³⁺ core UCNPs have a much narrow PSF but really low efficiency of generating fluorescence under low density excitation power. However, 8% Tm³⁺, 60% Yb³⁺ core-shell UCNPs have also an enhanced resolution (294.4 nm) due to the high-order nonlinearity of upconverting fluorescence with decent fluorescence generating efficiency under low power excitation density (5 kW/cm²).

Figure 3:

Laser scanning upconverting nonlinear image and saturated image of single 4% Tm³⁺, 20% Yb³⁺ core UCNPs and single 8% Tm³⁺, 60% Yb³⁺ core-shell UCNPs.

(A) 4% Tm³⁺, 20% Yb³⁺ core single UCNPs under nonlinear excitation (5×10³ W/cm²) and saturated excitation (100×10³ W/cm²), respectively. (B) 8% Tm³⁺, 60% Yb³⁺ core-shell single UCNPs under nonlinear excitation (5×10³ W/cm²) and saturated excitation (100×10³ W/cm²), respectively. Scale bar, 1 µm. The cross-section on the green dashed line in (A and B) are shown in (C and D). The fitted curves of nonlinear excitation (green) have a smaller FWHM than the saturated excitation ones (gray) due to different order of nonlinearity. (E and F) FWHM under different power density excitation of 4% Tm³⁺, 20% Yb³⁺ core UCNPs and 8% Tm³⁺, 60% Yb³⁺ core-shell UCNPs, respectively.

Using UCNPs with continuous wave (CW) IR excitation has similar merits as a multiphoton microscopy for deep tissue brain imaging, longer scattering length, and high-order nonlinearity. As the microscopy focus goes deep, the benefits of confocal detection diminish, and attention has to be paid to the detection of scattered light. Scattered light will spread to a volume that is much larger than when the excitation focus is deep below the scattering tissue. Large field detector-based LSM using nonlinear upconverting nanoprobe has the ability to realize deep tissue imaging, which is similar to femtosecond ultrafast laser-based multiphoton microscopy. Using upconverting microscopy with CW laser will make nonlinear deep tissue microscopy more cost-efficient and portable.

3 Conclusion

This work emphasizes the importance of controlling activator and sensitizer dopant concentrations to decrease excitation power for emitting the same upconverting luminescence intensity. We found the optimal activator and sensitizer concentration in single UCNPs that can increase upconverting luminescence and increase power dependence slopes. A shell coated on the surface to passivate the quenchers will shift the power dependence curve to lower excitation power. In addition, higher sensitizer concentration will help shift the power dependence curve to a lower excitation power. The core-shell structure will help improve luminescence intensity. This means that saturation intensity is more easily achieved under lower power excitation when activator concentration is increased.