Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy

Abstract

Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin.

Time-resolved fluorescence microscopy with phasor analysis for visualizing multicomponent topical drug distribution within human skin

“…In the previous approach 3,19 , cluster overlap between exogenous and endogenous species in the phasor space resulted in less accurate quantification and visualization of compound interactions at a point. However, considering the distance of a point in the phasor space to the centers of reference clusters now allows for a less biased calculation of compound contribution to a point in the treatment tissue sample.…”

“…Visualization of drug spatial distribution for single component topical drug. Previous studies have described and demonstrated the feasibility of FLIM along with phasor analysis for the sensitive visualization and quantification of a single component topical drug distribution 3,19 . Briefly, phasor analysis transforms all temporal fluorescence decay traces represented by pixels in a FLIM image into a pair of phasor coordinates (G, S) through a Fourier transform.…”

“…Previously, we reported using TPEF, FLIM, and phasor analysis to study the uptake of topically applied minocycline to the skin. Using FLIM and non-Euclidean phasor analysis allowed us to selectively visualize minocycline and extract its local distribution in the tissue 3,19 . In this study, we demonstrate the visualization of a multicompound drug formulation containing both minocycline and tazarotene.…”

Visualization of drug distribution of a topical minocycline gel in human facial skin

“…The non-Euclidean phasor analysis method has been described in a previously published article [17]. Briefly, the phasor analysis algorithm begins by transforming each temporal fluorescence decay trace into a pair of phasor coordinates (G, S), where G and S respectively correspond to the normalized real and imaginary components of the Fourier transform of the fluorescence decay traces evaluated at the laser repetition frequency.…”

Shedding light on topical drug uptake

“…To overcome these obstacles, pharmacokinetic studies of topical drug candidates have been recently carried out leveraging the nondestructive and noninvasive features of optical imaging methods such as multiphoton fluorescence microscopy, coherent Raman imaging and spectroscopy, and fluorescence lifetime imaging microscopy (FLIM) . These optical imaging methods show promising results for the visualization of drug distributions with molecularly specific contrast within skin at cellular and subcellular resolutions.…”