Methods for processing renal tissue samples for Single-Slice Dual-Mode optical correlation imaging
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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，基于两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。因此，发展单切片双模态光学关联成像技术，在单张切片上同时提供组织形态和多个目标蛋白的分布及表达，有助于更准确地描述肿瘤及其微环境。在样本量稀缺的肾脏病理检测中，该技术显得尤为重要：肾脏病理需要利用明场成像获取苏木素-伊红染色后组织和细胞的病理形态学信息，而利用荧光成像来获取多个目标蛋白的分布及表达情况则是肾脏免疫病理筛查的必检分子项目。本文重点研究了将苏木素-伊红染色和免疫荧光染色在同一张肾脏切片上实现的组织样本处理方法，对染色、褪色及复染的流程进行改良和效果对比，并探索将单切片双模态图像进行创新性融合。
Abstract:Bright-field imaging can provide cellular and histological morphological information, while fluorescence imaging can provide expression information of key proteins. Dual-modal correlation imaging based on both techniques is currently a common method for examining tissue samples in medical and scientific research. In clinical examination, however, correlation imaging between adjacent tissue slices is often used for observation. In such cases, both the tissue structure and the cellular level may be altered more or less, which is unfavorable when the sample volume is insufficient, the number of cells on the slices is limited, or precise point-to-point morphological information is required. Therefore, the development of single-slice dual-modal optical correlation imaging techniques which provides both tissue morphology and the distribution and expression of multiple target proteins on a single slice, can help to more accurately describe tumors and their microenvironment. This technique is particularly important in renal pathological testing where sample size is small. Renal pathology requires the use of bright-field imaging to obtain pathomorphological information of tissues and cells after hematoxylin-eosin staining, while the use of fluorescence imaging to obtain the distribution and expression of multiple target proteins is a mandatory molecular test for renal pathology screening. This paper focuses on the tissue sample processing methods that allow the coexistence of hematoxylin-eosin staining and immunofluorescence staining on the same renal slice. Improvements and comparative evaluations of the staining, de-colorizing and re-staining processes, as well as innovative fusion techniques for single-slice dual-modal imaging.
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图 1 小鼠肾脏组织石蜡切片HE染色后盐酸乙醇褪色的单切片双模态图。a、e、i为HE染色图；b−d是图a褪色后EDTA抗原修复的同一视野免疫荧光图；f−h是图b褪色后Tris-EDTA抗原修复的同一视野的免疫荧光图；j−l是图i褪色后柠檬酸抗原修复的同一视野免疫荧光图；m为盐酸乙醇褪色后不同抗原修复条件下的免疫荧光图像信噪比统计分析。标尺：50 μm。
Figure 1. The single slice bimodal images of the decolorization of hydrochloric acid ethanol after HE staining of paraffin slices of mouse renal tissue. a, e and i are HE staining images. b−d are the same visual field immunofluorescence images of EDTA antigen retrieval after de-colorizing in Fig. a. f−h are the immunofluorescence images of the same field of view of Tris-EDTA antigen retrieval after the decolorization of Fig. b. j−l are the same visual field immunofluorescence images of citric acid antigen retrieval after de-colorizing in Fig.i. m is the statistical analysis of the signal-to-noise ratio of immunofluorescence images under different antigen retrieval conditions after hydrochloric acid ethanol decolorization. Scale: 50 μm.
图 2 小鼠肾脏组织石蜡切片HE染色后冰醋酸-草酸褪色的单切片双模态图。a、e、i为HE染色图；b−d是图a褪色后EDTA抗原修复的同一视野免疫荧光图；f−h是图b褪色后Tris-EDTA抗原修复的同一视野的免疫荧光图；j−l是图i褪色后柠檬酸抗原修复的同一视野免疫荧光图；m为冰醋酸-草酸褪色后不同抗原修复条件下的免疫荧光图像信噪比统计分析。标尺：50 μm。
Figure 2. The single-slice bimodal images of glacial acetic acid-oxalic acid decolorization after HE staining of paraffin slices of mouse renal tissue. a, e and i are HE staining images, and b−d are the same visual field immunofluorescence images of EDTA antigen retrieval after de-colorizing in Fig.a. f−h are the immunofluorescence images of the same field of view of Tris-EDTA antigen retrieval after the decolorization of Fig. b. j−l are the same visual field immunofluorescence images of citric acid antigen retrieval after de-colorizing in Fig.i. m is the statistical analysis of the signal-to-noise ratio of immunofluorescence images under different antigen retrieval conditions after glacial acetic acid-oxalic acid decolorization. Scale: 50 μm.
图 3 小鼠肾脏组织石蜡切片HE染色后高锰酸钾-草酸褪色的单切片双模态图。a、e、i为HE染色图；b−d是图a褪色后EDTA抗原修复的同一视野免疫荧光图；f−h是图b褪色后Tris-EDTA抗原修复的同一视野的免疫荧光图；j−l是图i褪色后柠檬酸抗原修复的同一视野免疫荧光图；m为高锰酸钾-草酸褪色后不同抗原修复条件下的免疫荧光图像信噪比统计分析。标尺：50 μm。
Figure 3. The single-slice bimodal images of potassium permanganate-oxalic acid decolorization after HE staining of paraffin slices of mouse renal tissue. a, e, i are HE staining. b−d are the same visual field immunofluorescence images of EDTA antigen retrieval after de-colorizing in Fig.a. f−h are the immunofluorescence images of the same visual field of Tris-EDTA antigen retrieval after de-colorizing in Fig.b. j−l are the same visual field immunofluorescence images of citric acid antigen retrieval after de-colorizing in Fig.i. m is the statistical analysis of the signal-to-noise ratio of immunofluorescence images under different antigen repair conditions after potassium permanganate-oxalic acid decolorization. Scale: 50 μm.
图 4 小鼠肾脏组织HE染色后，使用冰醋酸和草酸褪色后并用EDTA修复所获得的单切片双模态图。a为60 ×镜下所采的HE染色；b为同视野下AQP-2和DAPI双标记的肾集合管免疫荧光图像；c为HE和免疫荧光融合图。
Figure 4. After HE staining, the mouse renal tissue was de-colorized with glacial acetic acid and oxalic acid and repaired with EDTA to obtain a single-slice bimodal image. a is HE staining obtained under a 60 × microscope. b is an immunofluorescence image of the renal collecting duct labeled with AQP-2 and DAPI in the same field of view. c is the fusion image of HE and immunofluorescence.
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