Bolognesi Maddalena M, Dall'Olio Lorenzo, Maerten Amy, Borghesi Simone, Castellani Gastone, Cattoretti Giorgio
Istituto di Bioimmagini e Fisiologia Molecolare - CNR, Milan, Italy.
National Biodiversity Future Center (NBFC), Palermo, Italy.
Biol Imaging. 2024 Oct 23;4:e10. doi: 10.1017/S2633903X24000138. eCollection 2024.
Hyperplexed in-situ targeted proteomics via antibody immunodetection (i.e., >15 markers) is changing how we classify cells and tissues. Differently from other high-dimensional single-cell assays (flow cytometry, single-cell RNA sequencing), the human eye is a necessary component in multiple procedural steps: image segmentation, signal thresholding, antibody validation, and iconographic rendering. Established methods complement the human image evaluation, but may carry undisclosed biases in such a new context, therefore we re-evaluate all the steps in hyperplexed proteomics. We found that the human eye can discriminate less than 64 out of 256 gray levels and has limitations in discriminating luminance levels in conventional histology images. Furthermore, only images containing visible signals are selected and eye-guided digital thresholding separates signal from noise. BRAQUE, a hyperplexed proteomic tool, can extract, in a marker-agnostic fashion, granular information from markers which have a very low signal-to-noise ratio and therefore are not visualized by traditional visual rendering. By analyzing a public human lymph node dataset, we also found unpredicted staining results by validated antibodies, which highlight the need to upgrade the definition of antibody specificity in hyperplexed immunostaining. Spatially hyperplexed methods upgrade and supplant traditional image-based analysis of tissue immunostaining, beyond the human eye contribution.
通过抗体免疫检测进行超多重原位靶向蛋白质组学（即>15种标志物）正在改变我们对细胞和组织的分类方式。与其他高维单细胞分析方法（流式细胞术、单细胞RNA测序）不同，人眼是多个操作步骤中的必要组成部分：图像分割、信号阈值设定、抗体验证和图像绘制。既定方法对人眼图像评估起到补充作用，但在这种新背景下可能存在未公开的偏差，因此我们重新评估了超多重蛋白质组学中的所有步骤。我们发现，人眼能够区分的灰度级少于256级中的64级，并且在区分传统组织学图像中的亮度水平方面存在局限性。此外，仅选择包含可见信号的图像，并且在人眼引导下进行数字阈值设定可将信号与噪声分离。BRAQUE是一种超多重蛋白质组学工具，它能够以与标志物无关的方式从信噪比非常低、因此无法通过传统视觉呈现可视化的标志物中提取颗粒信息。通过分析一个公开的人类淋巴结数据集，我们还发现经过验证的抗体出现了意外的染色结果，这凸显了在超多重免疫染色中升级抗体特异性定义的必要性。空间超多重方法超越了人眼的作用，升级并取代了传统的基于图像的组织免疫染色分析。
