De Giorgi V, Massi D, Sestini S, Cicchi R, Pavone F S, Lotti T
Department of Dermatology, University of Florence, Florence, Italy.
J Eur Acad Dermatol Venereol. 2009 Mar;23(3):314-6. doi: 10.1111/j.1468-3083.2008.03045.x. Epub 2009 Jan 14.
Two-photon excitation (TPE) fluorescence microscopy is a high-resolution laser-scanning imaging technique enabling deep imaging inside biological tissues. TPE microscopy has the triple advantage of offering high spatial resolution (250 nm radially, 800 nm axially), high penetration depth inside skin (200 mm ), and low photodamage effects. Further, cells and extracellular matrix intrinsically contain a variety of fluorescent molecules (NADH, tryptophan, keratins, melanin, elastin, cholecalciferol and others), so that biological tissues can be imaged by TPE microscopy without any exogenous probe. The time-resolved analysis of the fluorescence signal, known as fluorescence lifetime imaging microscopy (FLIM), is an additional non-invasive microscopy technique useful to characterize endogenous fluorescence species and their surrounding medium by measuring the mean lifetime of fluorescent emission. Finally, multispectral (MTPE) tissue imaging can also be used to identify different endogenous fluorescent species by measuring their two photon emission spectra. Those techniques offer functional information about the relative quantities of fluorescent molecules, which are correlated with tissue structure in physiological and pathological states.
We have decided to apply these three methods at the same time for cutaneous tumors in order to evaluate their possible future use.
We have analyzed a melanoma and a basal cell carcinoma, with their surrounding healthy skin, to evaluate any difference in healthy skin and neoplasia. The samples were excised during dermatological surgery, then cut, saving some healthy skin in both, to obtain a regular shape, allowing its positioning either with the skin surface parallel to the optical axis (horizontal optical sectioning), or perpendicular (vertical optical sectioning).
This first result demonstrates that FLIM is effective in discriminating healthy skin from MM, while MTPE is effective in discriminating healthy skin from BCC.
双光子激发（TPE）荧光显微镜是一种高分辨率激光扫描成像技术，能够对生物组织进行深度成像。TPE显微镜具有三重优势，即提供高空间分辨率（径向250纳米，轴向800纳米）、在皮肤内的高穿透深度（200毫米）以及低光损伤效应。此外，细胞和细胞外基质本身含有多种荧光分子（烟酰胺腺嘌呤二核苷酸、色氨酸、角蛋白、黑色素、弹性蛋白、胆钙化醇等），因此生物组织可以通过TPE显微镜进行成像而无需任何外源性探针。荧光信号的时间分辨分析，即荧光寿命成像显微镜（FLIM），是另一种非侵入性显微镜技术，通过测量荧光发射的平均寿命来表征内源性荧光物质及其周围介质。最后，多光谱（MTPE）组织成像也可用于通过测量其双光子发射光谱来识别不同的内源性荧光物质。这些技术提供了有关荧光分子相对数量的功能信息，这些信息与生理和病理状态下的组织结构相关。
我们决定同时将这三种方法应用于皮肤肿瘤，以评估它们未来可能的用途。
我们分析了一个黑色素瘤和一个基底细胞癌及其周围的健康皮肤，以评估健康皮肤和肿瘤之间的任何差异。样本在皮肤科手术期间切除，然后切割，在两者中都保留一些健康皮肤，以获得规则形状，使其能够以皮肤表面平行于光轴（水平光学切片）或垂直（垂直光学切片）的方式定位。
这一初步结果表明，FLIM在区分健康皮肤和黑色素瘤方面有效，而MTPE在区分健康皮肤和基底细胞癌方面有效。
