Development of an H&E on-block staining technique for collagen detection in cryo-fluorescence tomography imaging of frozen breast tissue samples.

IF 2.6 3区 综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES
PLoS ONE Pub Date : 2025-06-09 eCollection Date: 2025-01-01 DOI:10.1371/journal.pone.0324493
Erin P Snoddy, Tien T Tang, Natalie Fowlkes, Thomas Huynh, Kari J Brewer Savannah, Alejandro Contreras, Gregory Reece, Kristy K Brock
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Abstract

Hematoxylin and eosin (H&E) staining is widely considered to be the gold-standard diagnostic tool for histopathology evaluation. However, the fatty nature of some tissue types, such as breast tissue, presents challenges with cryo-sectioning, often resulting in artifacts that can make histopathologic interpretation and correlation with other imaging modalities virtually impossible. We present an optimized on-block H&E staining technique that improves contrast for identifying collagenous stroma during cryo-fluorescence tomography (CFT) sectioning. In this prospective study, we embedded four breast specimens with confirmed ligaments from a bilateral mastopexy in an optimal cutting temperature block. Two of the samples were processed on a CFT imager and stained with our on-block staining protocol. In this protocol, hematoxylin was applied to the block-face before being washed with deionized water. Eosin was then applied and washed with 95% ethanol. We then applied mounting medium and acquired images with a stereo-dissecting microscope and camera. Prior to staining, GFP fluorescence and white-light images were acquired with the CFT system to serve as a validation metric. The other two samples were sectioned on a standard cryostat and stained according to gold-standard H&E protocol. The resulting microscope slides were imaged with a digital slide scanner and viewed with Leica Imagescope software. An experienced pathologist evaluated both sets of images for qualitative comparisons. Pathologist evaluation confirmed that striations from on-block staining were qualitatively comparable with collagen tracks identified in gold-standard histology images. Furthermore, GFP images captured collagen autofluorescence, which aligned with the same structures identified by our on-block staining protocol. Our on-block staining technique shows comparable visualization of collagenous structures at the mesoscopic level for fresh breast tissue samples. This technique improves tissue contrast and region of interest selection for histology during CFT imaging for analysis of the stromal architecture of the breast.

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冷冻乳腺组织样本冷冻荧光断层成像中胶原蛋白检测的H&E块染色技术的发展。
苏木精和伊红(H&E)染色被广泛认为是组织病理学评估的金标准诊断工具。然而,某些组织类型的脂肪性质,如乳腺组织,对冷冻切片提出了挑战,通常会导致伪影,使组织病理学解释和与其他成像方式的相关性几乎不可能。我们提出了一种优化的块上H&E染色技术,提高了在冷冻荧光断层扫描(CFT)切片过程中识别胶原基质的对比度。在这项前瞻性研究中,我们将4个双侧乳房切除术后确认韧带的乳房标本置于最佳切割温度块中。两个样本在CFT成像仪上处理,并按照我们的块染色方案进行染色。在该方案中,在用去离子水洗涤之前,将苏木精涂在块面上。然后涂上伊红并用95%乙醇洗涤。然后我们使用安装介质和立体解剖显微镜和相机获取图像。染色前,用CFT系统获取GFP荧光和白光图像作为验证指标。另外两个样品在标准低温恒温器上切片,并根据金标准H&E方案染色。所得的显微镜载玻片用数字载玻片扫描仪成像,用徕卡Imagescope软件观察。一位经验丰富的病理学家评估了两组图像进行定性比较。病理学家评估证实,从块染色条纹定性可比胶原轨道鉴定的金标准组织学图像。此外,GFP图像捕获胶原蛋白自身荧光,与我们的块染色方案确定的相同结构一致。我们的块上染色技术在介观水平上显示了新鲜乳房组织样本的胶原结构的类似可视化。这项技术提高了组织对比和感兴趣的区域选择组织学在CFT成像分析乳腺间质结构。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
PLoS ONE
PLoS ONE 生物-生物学
CiteScore
6.20
自引率
5.40%
发文量
14242
审稿时长
3.7 months
期刊介绍: PLOS ONE is an international, peer-reviewed, open-access, online publication. PLOS ONE welcomes reports on primary research from any scientific discipline. It provides: * Open-access—freely accessible online, authors retain copyright * Fast publication times * Peer review by expert, practicing researchers * Post-publication tools to indicate quality and impact * Community-based dialogue on articles * Worldwide media coverage
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