Charité-Universitätsmedizin Berlin, Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Venerology and Allergology, Charitéplatz 1, 10117 Berlin, Germany.
J Biomed Opt. 2017 Sep 1;22(9):91503. doi: 10.1117/1.JBO.22.9.091503.
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.
自发荧光漂白描述了生物组织内源性荧光团在光照射下荧光强度的降低。自发荧光漂白的起源尚未完全阐明。在皮肤中，各种内源性荧光团的空间分布在皮肤层内有所不同。大多数内源性荧光团在紫外线和短可见光波长范围内被激发，只有少数几种，如卟啉（红色）和黑色素（近红外），在较长波长处被激发。因此，皮肤的激发波长和深度依赖性辐照将激发不同的荧光团，这可能会影响漂白特性。使用 325、473、633 和 785nm 激发，在 150?? m 的深度范围内从皮肤表面到真皮，对猪耳朵皮肤进行了离体测量，测量了自发荧光漂白。共聚焦拉曼显微镜用于实现测量的足够空间分辨率。在 21 次连续 15 秒采集的过程中，测量了荧光曲线下的面积。在所有情况下，漂白都遵循双指数衰减函数，通过非线性回归进行近似。结果表明，漂白可用于提高所有应用的激发波长和皮肤深度的拉曼光谱中的信噪比。
