用于单切片双模态光学关联成像的肾脏组织样本处理方法  

Methods for processing renal tissue samples for single-slice dual-mode optical correlation imaging

作　　者：高歌 郭晓光 吴俊楠 陈海龙 史冰 黄振立 GAO Ge;GUO Xiao-guang;WU Jun-nan;CHEN Hai-long;SHI Bing;HUANG Zhen-li(Key Laboratory of Biomedical Engineering of Hainan Province,School of Biomedical Engineering,Hainan University,Haikou 570100,China;School of Computer Science and Technology,Hainan University,Haikou 570100,China)

机构地区：[1]海南省生物医学工程重点实验室海南大学生物医学工程学院,海南海口570100 [2]海南大学计算机科学与技术学院,海南海口570100

出　　处：《中国光学（中英文）》2024年第5期1227-1235,共9页Chinese Optics

基　　金：海口市重点科技计划项目(No.2021-016)。

摘　　要：明场成像能够提供细胞或组织的形态学信息,荧光成像可以获取关键蛋白的表达信息,基于二者的双模态关联成像是目前医学和科研中常用的组织样本检查方式。然而,在临床检查时通常利用基于邻近切片之间的关联成像进行观察。此时,组织结构和细胞层次均会有或多或少的改变,这在样本量不足、切片上的细胞有限或需要获得点对点精准形态学信息的情景下显得十分不利。本研究提出了一种在单张组织切片中实现苏木素-伊红染色和免疫荧光染色的样本处理方法,用于双模态成像技术。重点优化了褪色处理和免疫荧光复染方案,比较了三种褪色方案(盐酸乙醇、冰醋酸-草酸和高锰酸钾-草酸)以及三种抗原修复方案(EDTA、Tris-EDTA和柠檬酸)。通过对不同条件下获取的图像信噪比进行对比分析,发现经冰醋酸-草酸褪色结合EDTA抗原修复的免疫荧光图像质量最佳。此外,还实现了明场与荧光图像的融合,从而在单张切片上展示更完整的组织形态和免疫信息。Bright-field imaging can provide cellular and histological morphological information,while fluorescence imaging can provide expression information of key proteins.Dual-mode correlation imaging that combines both techniques is used for examining tissue samples in medical and scientific research.However,in clinical,correlation imaging often relies on adjacent tissue sections for observation.This can result in inconsistencies at both the tissue structure and the cellular level,which is problematic when the sample volume is limited,the number of cells on the slices is sparse,or precise point-to-point morphological information is required.In this study,we present a sample preparation method that enables both Hematoxylin and Eosin(H&E)staining and immunofluorescence staining on a single tissue section,enabling dual-mode imaging.We focused on optimizing the decolorizing process and the immunofluorescence restaining protocol,comparing three decolorization methods(hydrochloric acid ethanol,acetic acid-oxalic acid,and potassium permanganate-oxalic acid)and three antigen retrieval methods(EDTA,Tris-EDTA,and citric acid).By analyzing the signal-to-noise ratios(SNR)of images obtained under different conditions,we found that the combination of acetic acid-oxalic acid decolorization with EDTA antigen retrieval produced the best quality immunofluorescence images.Additionally,we successfully fused brightfield and fluorescence images,providing more comprehensive tissue morphology and immunological information on a single section.

关 键 词：双模态关联成像 免疫荧光成像 褪色处理 抗原修复 关联成像分析 

分 类 号：TP394.1[自动化与计算机技术—计算机应用技术] TH691.9[自动化与计算机技术—计算机科学与技术]