Institute of Biomedical Engineering, The Second Clinical Medical College (Shenzhen People's Hospital), Jinan University, Shenzhen 518020, P. R. China.
The First Affiliated Hospital, Jinan University, Guangzhou 510632, P. R. China.
Anal Chem. 2021 Apr 20;93(15):6195-6204. doi: 10.1021/acs.analchem.1c00390. Epub 2021 Apr 7.
Single-molecule (SM) quantification has become a powerful analytical technique in the fields of physics, chemistry, and biology. SM imaging, especially with super-resolution (SR) techniques, has dramatically facilitated the study of individual molecules that may function as disease-related biomarkers. Although multiple properties can be used for quantitative imaging analysis, counting may be the simplest and most direct way. Consequently, how to utilize the greater spatial resolution to overcome undercounting or overcounting errors in certain conditions shows promising potential to unravel intracellular mechanisms of isolated biomolecules. From this perspective, we present an absolute quantification approach, termed crucial connected-component entropy (CCCE), with subresolution accuracy for the SR SM detection platform without the need for prior knowledge of calibration, and a cross-validation analytical pipeline based on SM profiling for nanoscale performance assessments. Considering its high efficiency, accuracy, and robustness for routine SM quantification compared with commonly used strategies, we believe that this protocol will indubitably find wide applications in biochemistry research, drug discovery, and clinical diagnostics, especially molecular diagnostics.
单分子（SM）定量分析已经成为物理、化学和生物学领域的一种强大分析技术。SM 成像，特别是具有超分辨率（SR）技术的 SM 成像，极大地促进了对可能作为疾病相关生物标志物的单个分子的研究。虽然可以使用多种特性进行定量成像分析，但计数可能是最简单和最直接的方法。因此，如何利用更高的空间分辨率克服在某些条件下的欠计数或过计数误差，为揭示分离生物分子的细胞内机制展示了很有前途的潜力。从这个角度来看，我们提出了一种绝对定量方法，称为关键连通分量熵（CCCE），该方法具有亚分辨率精度，适用于 SR SM 检测平台，而无需事先进行校准的知识，并且基于 SM 分析的交叉验证分析管道可用于纳米级性能评估。考虑到与常用策略相比，该方法在常规 SM 定量方面具有高效、准确和稳健的特点，我们相信该方案将在生物化学研究、药物发现和临床诊断，特别是分子诊断中得到广泛应用。
