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。
Abstract: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.
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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。标尺为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 field of view 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 field of view 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。标尺为50 μm
Figure 2. The single-slice bimodal images of glacial acetic acid and 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 field of view 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 field of view 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。标尺为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 field of view 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 de-colorizing in Fig.(b). (j)~(l) are the same field of view 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. The single-slice bimodal images of the mouse renal tissue de-colorized with glacial acetic acid and oxalic acid and repaired with EDTA, after HE staining. (a) is HE staining image 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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