The Imaging Technique Of Two-photon Excitation Fluorescence Microscopy In The Diagnosis Of Gallbladder Cancers

Aim The gold standard for diagnosis of gallbladder cancers is hematoxylin-eosin(H-E) staining histopathology. However, the conventional technology has several disadvantages, including relatively time-consuming, environmental pollution and staining problems. The novel imaging technique of two-photon excited fluorescence(TPEF) microscopy, based on autofluorescence signal from cells, can provide detailed information on tissue architecture and cellular morphology in unstained histological sections to generate subcellularresolution images from tissue directly. In this paper, we used TPEF microscopy to image microstructure of human normal gallbladder and three types of differentiated carcinomas to investigate the morphological changes of gallbladder cancers of tissue structure, cell, cytoplasm, and nucleus without hematoxylin and eosin(H&E) staining, and conduct blinded trial, to evaluate the feasibility and significance of using TPEF microscopy imaging techniques for optical diagnosis of gallbladder cancers.Methods: Firstly, the histological sections of human normal gallbladder and three types of differentiated carcinomas underwent deparaffinating and TPEF microscope imaging, and then went through H-E staining process and optical microscope. The histopathology of TPEF images and H-E staining images were compared and analyzed. Secondly, a pilot study was performed to establish the optical diagnostic features of TPEF imaging for gallbladder cancers by comparing the histological changes and the area differences of the nucleus and the nucleolus in three types of differentiated carcinomas and normal gallbladder. Finally, a blinded study wasconducted to test the accuracy of TPEF optical diagnosis of 28 histological sections of normal gallbladder and three types of different differentiated gallbladder cancers.Result: It displayed that TPEF microscopy can well image the stratified normal gallbladder tissue, including the mucosa, the muscularis, and the serosa. The typical cancer cell, characterized by cellular and nuclear pleomorphism, enlarged nuclei, and augmented nucleolus, can be identified in histological sections without H-E staining as well. Based on morphological differences in the organization, TPEF microscopy imaging can not only identify the histological and cytological features in well, moderately and poorly differentiated gallbladder cancer, but also differentiate the differences of the nuclear and nucleolar area between gallbladder cancers and the normal gallbladder, which were comparable to H-E stained images. In the blinded trial, the accuracy of optical diagnosis of TPEF imaging was 96.4%.Conclusion: The studies demonstrated that TPEF microcopy has the ability to differentiate gallbladder cancers from normal gallbladder, and further distinguish differentiated carcinomas based on the morphological alteration, which were kept in histological sections without H&E staining. The results from TPEF images were comparable to H&E images, even more superior to H&E in terms of locating the position of nuclei and nucleolus. Our study provided the basic data to develop two-photon imaging for performing retrospective studies of gallbladder cancer. With the development of miniaturization and integration, the label-free and nonpolluting TPEF imaging may be an assistive tool for pathological diagnoses to provide richer information for H&E staining and diagnosed more accurately.