This study tests the use of a constrained nonnegative matrix factorization (NMF) algorithm to explore the comparatively new field of chemometric microscopy to support tissue diagnosis. The algorithm can extract the spectral signature and the absolute concentration map of endogenous fluorophores from wide-field microscopic images. The resultant data distinguished normal and fibrous calvarial tissues, based on the changes in their spectral signatures. The absolute concentration map of endogenous fluorophores, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and lipofuscin were derived from microscopic images and compared with the fluorescence from pure fluorophores. While the absolute concentration of NADH increased, the same of FAD and lipofuscin decreased from a normal to fibrous calvarial condition. An increase in the optical redox ratio, possibly due to the metabolic changes during the development of fibrosis, was observed. Differentiating tissue types using the absolute concentration map was found to be considerably more precise than that achievable with relative concentration. The quantification of fluorophores with reference to the absolute concentration map can eliminate uncertainties due to system responses or measurement details, thereby generating more biologically apposite data. Wide-field microscopy augmented with a constrained NMF algorithm could emerge as an advanced diagnostic tool, potentially heralding the emergence of chemometric microscopy.

中文翻译：

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用于组织诊断的宽视场荧光显微图像的半监督非负矩阵分解

本研究测试了使用受约束的非负矩阵分解 (NMF) 算法来探索化学计量显微镜这一相对较新的领域以支持组织诊断。该算法可以从广域显微图像中提取内源性荧光团的光谱特征和绝对浓度图。结果数据根据光谱特征的变化区分了正常和纤维颅骨组织。内源性荧光团、烟酰胺腺嘌呤二核苷酸 (NADH)、黄素腺嘌呤二核苷酸 (FAD) 和脂褐质的绝对浓度图来自显微图像，并与纯荧光团的荧光进行了比较。虽然 NADH 的绝对浓度增加，但 FAD 和脂褐质的绝对浓度从正常颅骨状态下降到纤维颅骨状态。观察到光学氧化还原比的增加，可能是由于纤维化发展过程中的代谢变化。发现使用绝对浓度图区分组织类型比使用相对浓度可实现的组织类型要精确得多。参考绝对浓度图对荧光团进行量化可以消除由于系统响应或测量细节引起的不确定性，从而产生更符合生物学的数据。使用受限 NMF 算法增强的广域显微镜可能会成为一种先进的诊断工具，可能预示着化学计量显微镜的出现。发现使用绝对浓度图区分组织类型比使用相对浓度可实现的组织类型要精确得多。参考绝对浓度图对荧光团进行量化可以消除由于系统响应或测量细节引起的不确定性，从而产生更符合生物学的数据。使用受限 NMF 算法增强的广域显微镜可能会成为一种先进的诊断工具，可能预示着化学计量显微镜的出现。发现使用绝对浓度图区分组织类型比使用相对浓度可实现的组织类型要精确得多。参考绝对浓度图对荧光团进行量化可以消除由于系统响应或测量细节引起的不确定性，从而产生更符合生物学的数据。使用受限 NMF 算法增强的广域显微镜可能会成为一种先进的诊断工具，可能预示着化学计量显微镜的出现。