Zheng Wei, Wu Yicong, Li Dong, Qu Jianan Y
Hong Kong University of Science and Technology, Department of Electronic and Computer Engineering, Kowloon, Hong Kong.
J Biomed Opt. 2008 Sep-Oct;13(5):054010. doi: 10.1117/1.2975866.
We instrumented a combined fluorescence spectroscopy and imaging system to characterize the single- and two-photon excited autofluorescence in epithelial tissue. Single-photon fluorescence (SPF) are compared with two-photon fluorescence (TPF) measured at the same location in epithelial tissue. It was found that the SPF and TPF signals excited at corresponding wavelengths are similar in nonkeratinized epithelium, but the SPF and TPF spectra in the keratinized epithelium and the stromal layer are significant different. Specifically, the comparison of SPF signals with TPF signals in keratinized epithelial and stromal layers shows that TPF spectral peaks always have about 15-nm redshift with respect to SPF signals, and the TPF spectra are broader than SPF spectra. The results were generally consistent with the SPF and TPF measurements of pure nicotinamide adenine dinucleotide, flavin adenine dinucleotide, keratin and collagen, the major fluorophores in epithelium and stroma, respectively. The double peak structure of TPF spectra measured from keratinized layer suggests that there may be an unknown fluorophore responsible for the spectral peak in the long wavelength region. Furthermore, the TPF signals excited in a wide range of wavelengths provide accurate information on epithelial structure, which is an important advantage of TPF over SPF spectroscopy in the application for the diagnosis of tissue pathology.
我们设计了一种结合荧光光谱和成像的系统，以表征上皮组织中的单光子和双光子激发自体荧光。将上皮组织中同一位置测量的单光子荧光（SPF）与双光子荧光（TPF）进行比较。结果发现，在非角质化上皮中，对应波长激发的SPF和TPF信号相似，但在角质化上皮和基质层中，SPF和TPF光谱存在显著差异。具体而言，角质化上皮层和基质层中SPF信号与TPF信号的比较表明，TPF光谱峰相对于SPF信号总是有大约15纳米的红移，并且TPF光谱比SPF光谱更宽。这些结果与分别对上皮和基质中的主要荧光团——纯烟酰胺腺嘌呤二核苷酸、黄素腺嘌呤二核苷酸、角蛋白和胶原蛋白进行的SPF和TPF测量结果基本一致。从角质化层测量的TPF光谱的双峰结构表明，可能存在一种未知的荧光团导致长波长区域的光谱峰。此外，在很宽波长范围内激发的TPF信号提供了关于上皮结构的准确信息，这是TPF在组织病理学诊断应用中相对于SPF光谱的一个重要优势。
