There are few non-animal options for assessing nanoparticle-containing cosmetics’ exposure and toxicity, such as sunscreens. The aim of this study was to see how well timecorrelated single photon counting (TCSPC) could be used to track zinc oxide nanoparticles (ZnO-NP) and the metabolic state of volunteer skin at the same time.
We used advanced TCSPC to distinguish the fluorescence lifetime signatures of endogenous fluorophore signals (i.e. nicotinamide adenine dinucleotide dinucleotide phosphate, NAD(P)H, and keratin) and the ZnO-NP signal to establish ZnO-NP penetration profiles and NAD(P)H changes in subjects with altered barrier function, such as tape-stripped skin and ps
In any community, we found no ZnO-NP penetration into viable human skin. After 4 and 2 hours of therapy, the ZnO-NP signal was significantly increased (p 0.01) on the surface of tape-stripped and lesional skin, respectively. When tape-stripped viable epidermis was handled for 4 hours with ZnO-NP, the free NAD(P)H signal increased significantly compared to the vehicle power. The lesional analysis found no major changes in NAD(P)H.
Single photon counting systems overview
TCSPC (time-correlated single photon counting) is a popular technique for calculating time-domain fluorescence decays. Single photon events are observed in general, and the time of arrival is associated to the laser pulse used to excite the sample. This process can be replicated several times using a pulsed laser with a high repetition rate, resulting in a photon distribution over time and spatial coordinates. The operation theory is further explained in Fig. 1.
Time-correlated single photon counting (TCSPC) measurements are shown in Figure 1 as an example of how they operate. A high-repetition-rate pulsed laser source is used to excite the sample. A high-gain photomiltiplier measures photons produced by the sample, and the time with respect to the excitation pulse is measured. A histogram of the photon distribution over time is constructed by counting several events. (Reproduced with permission from Becker, The bh TCSPC Handbook, 6th ed., 2014, p. 69 – Link)
The arrival time of the photon and the coordinates of the laser beam in the scan region are used to create a 3-dimensional matrix on the stage of a laser scanning microscope, in which not only the fluorescence intensity, but also a fluorescence decay curve is stored for each pixel of the scanned image.
For fluorescence analysis, a time-correlated photon counter is proposed and evaluated as an ultralow level light detection module. The time-correlated photon counter counts photons reliably by using a silicon photomultiplier as a photon counting sensor in combination with a Poisson statistics algorithm and a double time windows technique. The time-correlated photon counter can record the arrival time of very weak light signals and is consistent with the time-correlated single photon counting technique. The strength and lifetime of fluorescein isothiocyanate were calculated using this low-cost and compact instrument, which yielded a limit of detection of 16 pg/ml with a wide linear dynamic range of 2.86 pg/ml to 0.5 g/ml and a lifetime of 3.758 ns, which agrees well with the findings of a sophisticated commercial fluorescence analysis instrument. The time-correlated photon counter could be useful in point-of-care research, for example.
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677102 (11 October 2007); https://doi.org/10.1117/12.735546. Wolfgang Becker and Axel Bergmann, “Multi-dimensional time-correlated single-photon counting,” Proc. SPIE 6771, Advanced Photon Counting Techniques II, 677102 (11 October 2007).