Depth-dependent autofluorescence photobleaching using 325, 473, 633, and 785 nm of porcine ear skin ex vivo

Abstract

Autofluorescence photobleaching describes the decrease of fluorescence intensity of endogenous fluorophores in biological tissue upon light irradiation. The origin of autofluorescence photobleaching is not fully understood. In the skin, the spatial distribution of various endogenous fluorophores varies within the skin layers. Most endogenous fluorophores are excited in the ultraviolet and short visible wavelength range, and only a few, such as porphyrins (red) and melanin (near-infrared), are excited at longer wavelengths. The excitation wavelength- and depth-dependent irradiation of skin will therefore excite different fluorophores, which will likely influence the photobleaching characteristics. The autofluorescence photobleaching of porcine ear skin has been measured ex vivo using 325, 473, 633, and 785 nm excitation at different skin depths from the surface to the dermis at 150 ? ? m . Confocal Raman microscopes were used to achieve sufficient spatial resolution of the measurements. The autofluorescence area under the curve was measured for 21 consecutive acquisitions of 15 s. In all cases, the photobleaching follows a two-exponential decay function approximated by nonlinear regression. The results show that photobleaching can be applied to improve the signal-to-noise ratio in Raman spectroscopy for all of the applied excitation wavelengths and skin depths.

Stratum corneum occlusion induces water transformation towards lower bonding state: a molecular level in vivo study by confocal Raman microspectroscopy

“…2°C [29] and the low amount of free radicals generated [30,31]. Depth-and dose-dependent fluorescence photobleaching does not influence the Raman peak intensities [32], which ensures comparable results. Raman spectra in the FP and HWN regions were acquired at the same positions at 2 µm increments between the position of approx.…”

Human skin in vivo has a higher skin barrier function than porcine skin ex vivo—comprehensive Raman microscopic study of the stratum corneum

“…Figure exemplarily shows Raman spectra of human SC in vivo in the fingerprint and HWN regions. The effect of fluorescence photobleaching in the SC does not influence the Raman peak intensities , which ensures comparable Raman results. The spatial depth resolution was ≤5 μm and the spectral resolution was 2 cm −1 , respectively .…”

In vivo confocal Raman microscopic determination of depth profiles of the stratum corneum lipid organization influenced by application of various oils

“…The utilized doses of excitation light (1.1 J/cm 2 for 785 nm and 0.2 J/ cm 2 for 671 nm excitation) can be considered safe for human skin concerning local temperature increase (maximal 2 C) [52] and with regard to light-induced free radical generation [53,54]. The dose-dependent fluorescence photobleaching in the SC, without influence on Raman peak intensities was recently reported [55].…”