十二指肠胃素瘤（DGASTs）是神经内分泌肿瘤，发生在十二指肠黏膜下层，并产生胃素激素。由于这些肿瘤体积小且易于在十二指肠中扩散，因此DGASTs的手术切除比较困难。经内镜黏膜切除DGASTs是一种越来越受欢迎的治疗方法，由于其低并发症率，但其病理学阴性边缘率低。多光子显微镜可通过内源性荧光（自发光[AF]）通过双光子激发荧光（2PEF）产生的对比度捕获生物组织的高分辨率图像。第二谐波产生是多光子显微镜产生图像对比度的另一种常见方法，主要出现在生物样本中如胶原结构中的光散射现象。一些有助于AF的分子由于与癌症过程相关的过程（例如代谢变化、氧化应激和血管生成）而丰富起来。使用MPM对12个单独的固定在福尔马林和石蜡中嵌入的十二指肠胃素瘤切片的患者样本进行成像，其中包括一个第二谐波产生通道和四个2PEF通道。每个2PEF通道的激发和发射谱被调整，以捕获由具有已知荧光光谱和与癌组织中生理变化有关的荧光物质主导的信号。我们发现，与十二指肠相邻组织相比，DGASTs区域生成的2PEF通道信号相对丰富的差异显著。从MPM图像中提取纹理特征生成的数据被用于线性判别分析模型，以在主成分分析（PCA）之前和之后为肿瘤与所有其他组织类型创建分类器。PCA提高了分类器的准确性，并减少了实现最大准确性所需的特征数量。在PCA之后的线性判别分类器将肿瘤与其他组织类型区分开来，准确率为90.6％-93.8％。这些结果表明，多光子显微镜2PEF和SHG成像是一种有前途的无标记方法，可区分DGASTs和正常十二指肠组织，这对于将来的内窥镜MPM切除边缘的在体评估具有影响力。©2022 Wiley Periodicals LLC。

Duodenal gastrinomas (DGASTs) are neuroendocrine tumors that develop in the submucosa of the duodenum and produce the hormone gastrin. Surgical resection of DGASTs is complicated by the small size of these tumors and the tendency for them to develop diffusely in the duodenum. Endoscopic mucosal resection of DGASTs is an increasingly popular method for treating this disease due to its low complication rate but suffers from poor rates of pathologically negative margins. Multiphoton microscopy can capture high-resolution images of biological tissue with contrast generated from endogenous fluorescence (autofluorescence [AF]) through two-photon excited fluorescence (2PEF). Second harmonic generation is another popular method of generating image contrast with multiphoton microscopy (MPM) and is a light-scattering phenomenon that occurs predominantly from structures such as collagen in biological samples. Some molecules that contribute to AF change in abundance from processes related to the cancer disease process (e.g., metabolic changes, oxidative stress, and angiogenesis).MPM was used to image 12 separate patient samples of formalin-fixed and paraffin-embedded duodenal gastrinoma slides with a second-harmonic generation (SHG) channel and four 2PEF channels. The excitation and emission profiles of each 2PEF channel were tuned to capture signal dominated by distinct fluorophores with well-characterized fluorescent spectra and known connections to the physiologic changes that arise in cancerous tissue.We found that there was a significant difference in the relative abundance of signal generated in the 2PEF channels for regions of DGASTs in comparison to the neighboring tissues of the duodenum. Data generated from texture feature extraction of the MPM images were used in linear discriminant analysis models to create classifiers for tumor versus all other tissue types before and after principal component analysis (PCA). PCA improved the classifier accuracy and reduced the number of features required to achieve maximum accuracy. The linear discriminant classifier after PCA distinguished between tumor and other tissue types with an accuracy of 90.6%-93.8%.These results suggest that multiphoton microscopy 2PEF and SHG imaging is a promising label-free method for discriminating between DGASTs and normal duodenal tissue which has implications for future applications of in vivo assessment of resection margins with endoscopic MPM.© 2022 Wiley Periodicals LLC.