Dey Simli, Maiti Sudipta
Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India.
J Biosci. 2018 Jul;43(3):447-454.
Single-molecule photobleaching (smPB) technique is a powerful tool for characterizing molecular assemblies. It can provide a direct measure of the number of monomers constituting a given oligomeric particle and generate the oligomer size distribution in a specimen. A major current application of this technique is in understanding protein aggregation, which is linked to many incurable diseases. Quantitative measurement of the size distribution of an aggregating protein in a physiological solution remains a difficult task, since techniques such as dynamic light scattering or fluorescence correlation spectroscopy (FCS) can provide an average size, but cannot accurately resolve the underlying size distribution. Here we describe the smPB method as implemented on a home-built total internal reflection fluorescence microscope (TIRF). We first describe the construction of a TIRF microscope, and then demonstrate the power of smPB by characterizing a solution of Amylin (hIAPP) oligomers, a 37-residue peptide whose aggregation is associated with Type II diabetes. We compare our results with FCS data obtained from the same specimen, and discuss the advantages and disadvantages of the two techniques.
单分子光漂白（smPB）技术是表征分子聚集体的有力工具。它可以直接测量构成给定寡聚体颗粒的单体数量，并生成样品中的寡聚体尺寸分布。该技术目前的一个主要应用是理解与许多不治之症相关的蛋白质聚集。在生理溶液中对聚集蛋白的尺寸分布进行定量测量仍然是一项艰巨的任务，因为诸如动态光散射或荧光相关光谱（FCS）等技术可以提供平均尺寸，但无法准确解析潜在的尺寸分布。在这里，我们描述了在自制的全内反射荧光显微镜（TIRF）上实现的smPB方法。我们首先描述TIRF显微镜的构建，然后通过表征胰淀素（hIAPP）寡聚体溶液来展示smPB的能力，胰淀素是一种37个残基的肽，其聚集与II型糖尿病有关。我们将我们的结果与从同一标本获得的FCS数据进行比较，并讨论这两种技术的优缺点。
