Quasi-equilibrium state based quantification of biological macromolecules in single-molecule localization microscopy

IF 2.4 3区 化学 Q3 CHEMISTRY, ANALYTICAL Methods and Applications in Fluorescence Pub Date : 2023-07-18 DOI:10.1101/2023.07.17.549270
Xuecheng Chen, Yaqian Li, Xiaowei Li, Jielin Sun, D. Czajkowsky, Zhifeng Shao
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引用次数: 1

Abstract

The stoichiometry of molecular components within supramolecular biological complexes is often an important property to understand their biological functioning, particularly within their native environment. While there are well established methods to determine stoichiometry in vitro, it is presently challenging to precisely quantify this property in vivo, especially with single molecule resolution that is needed for the characterization stoichiometry heterogeneity. Previous work has shown that optical microscopy can provide some information to this end, but it can be challenging to obtain highly precise measurements at higher densities of fluorophores. Here we provide a simple approach using already established procedures in single-molecule localization microscopy (SMLM) to enable precise quantification of stoichiometry within individual complexes regardless of the density of fluorophores. We show that by focusing on the number of fluorophore detections accumulated during the quasi equilibrium-state of this process, this method yields a 50-fold improvement in precision over values obtained from images with higher densities of active fluorophores. Further, we show that our method yields more correct estimates of stoichiometry with nuclear pore complexes and is easily adaptable to quantify the DNA content with nanodomains of chromatin within individual chromosomes inside cells. Thus, we envision that this straightforward method may become a common approach by which SMLM can be routinely employed for the accurate quantification of subunit stoichiometry within individual complexes within cells.
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单分子定位显微镜中基于准平衡态的生物大分子定量
超分子生物复合物中分子组分的化学计量学通常是了解其生物功能的重要性质,特别是在其天然环境中。虽然有很好的方法来确定体外化学计量,但目前在体内精确量化这一特性是具有挑战性的,特别是在表征化学计量异质性所需的单分子分辨率下。以前的工作已经表明,光学显微镜可以为此提供一些信息,但是在更高密度的荧光团下获得高度精确的测量可能具有挑战性。在这里,我们提供了一种简单的方法,使用单分子定位显微镜(SMLM)中已经建立的程序,无论荧光团的密度如何,都可以精确定量单个复合物内的化学计量学。我们表明,通过关注在该过程的准平衡状态中积累的荧光团检测的数量,该方法产生的精度比从具有较高活性荧光团密度的图像中获得的值提高50倍。此外,我们表明,我们的方法对核孔复合物的化学计量学产生了更正确的估计,并且很容易适用于定量细胞内单个染色体内染色质纳米结构域的DNA含量。因此,我们设想这种简单的方法可能成为一种常见的方法,通过这种方法,SMLM可以常规地用于精确定量细胞内单个复合物内的亚基化学计量。
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来源期刊
Methods and Applications in Fluorescence
Methods and Applications in Fluorescence CHEMISTRY, ANALYTICALCHEMISTRY, PHYSICAL&n-CHEMISTRY, PHYSICAL
CiteScore
6.20
自引率
3.10%
发文量
60
期刊介绍: Methods and Applications in Fluorescence focuses on new developments in fluorescence spectroscopy, imaging, microscopy, fluorescent probes, labels and (nano)materials. It will feature both methods and advanced (bio)applications and accepts original research articles, reviews and technical notes.
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