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Biophysics Reports 2022, 8(2): 100-118 https://doi.org/10.52601/bpr.2022.210047
Review | Open Access | Issue | Published: 25 March 2022

Principles of fluorescence correlation spectroscopy applied to studies of biomolecular liquid–liquid phase separation

Show Author's Information Zhulou Wang1,  Huizhi Zhang1,  Lin Jian1,  Bo Ding3,  Keying Huang3,  Wolun Zhang3,  Qian Xiao1,  Shaohui Huang1,2( )
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
School of Life Sciences, University of Chinese Academy of Sciences, Beijing 101499, China
LightEdge Technologies Limited, Zhongshan, Guangdong 528403, China
Keywords:
Diffusion coefficient, Binding affinity, Liquid–liquid phase separation, Fluorescence correlation spectroscopy, Hydrodynamic radius
Cite this article:
Wang Z, Zhang H, Jian L, et al. Principles of fluorescence correlation spectroscopy applied to studies of biomolecular liquid–liquid phase separation. Biophysics Reports, 2022, 8(2): 100-118. https://doi.org/10.52601/bpr.2022.210047
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Abstract Full text About this article

Fluorescence correlation spectroscopy (FCS) investigates the temporal relationship of fluctuating fluorescence signals reflecting underlying molecular processes occurring in a solution sample or a single live cell. This review article introduces the principles of two basic and most used FCS techniques: fluorescence auto-correlation spectroscopy (FACS) and fluorescence cross-correlation spectroscopy (FCCS). Combined, FACS and FCCS techniques can quantitatively analyze multiple properties of molecule or nanoparticle samples, including molar concentration, diffusion coefficient and hydrodynamic radius, homo- or hetero-interaction, fluorescence brightness, etc. Not surprisingly, FCS techniques have long been used to investigate molecular mechanisms of biomolecular phase separation, first in the lipid bilayer and more recently in cell cytosol and nucleoplasm. The latter applications are especially exciting since a whole new class of membraneless cellular organelles have been discovered, which are proposed to be results of biomolecule liquid-liquid phase separation (LLPS). LLPS research can benefit significantly from the multifunctionality and single-molecule sensitivity of a variety of FCS techniques, particularly for live-cell studies. This review illustrates how FACS and FCCS techniques can be used to investigate multiple aspects of the molecular mechanisms of LLPS, and summarizes FCS applications to LLPS research in vivo and in vitro.


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About this article

Principles of fluorescence correlation spectroscopy applied to studies of biomolecular liquid–liquid phase separation

Show Author's information Zhulou Wang1, Huizhi Zhang1, Lin Jian1, Bo Ding3, Keying Huang3, Wolun Zhang3, Qian Xiao1, Shaohui Huang1,2( )
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
School of Life Sciences, University of Chinese Academy of Sciences, Beijing 101499, China
LightEdge Technologies Limited, Zhongshan, Guangdong 528403, China

Abstract

Fluorescence correlation spectroscopy (FCS) investigates the temporal relationship of fluctuating fluorescence signals reflecting underlying molecular processes occurring in a solution sample or a single live cell. This review article introduces the principles of two basic and most used FCS techniques: fluorescence auto-correlation spectroscopy (FACS) and fluorescence cross-correlation spectroscopy (FCCS). Combined, FACS and FCCS techniques can quantitatively analyze multiple properties of molecule or nanoparticle samples, including molar concentration, diffusion coefficient and hydrodynamic radius, homo- or hetero-interaction, fluorescence brightness, etc. Not surprisingly, FCS techniques have long been used to investigate molecular mechanisms of biomolecular phase separation, first in the lipid bilayer and more recently in cell cytosol and nucleoplasm. The latter applications are especially exciting since a whole new class of membraneless cellular organelles have been discovered, which are proposed to be results of biomolecule liquid-liquid phase separation (LLPS). LLPS research can benefit significantly from the multifunctionality and single-molecule sensitivity of a variety of FCS techniques, particularly for live-cell studies. This review illustrates how FACS and FCCS techniques can be used to investigate multiple aspects of the molecular mechanisms of LLPS, and summarizes FCS applications to LLPS research in vivo and in vitro.

Keywords: Diffusion coefficient, Binding affinity, Liquid–liquid phase separation, Fluorescence correlation spectroscopy, Hydrodynamic radius

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Received: 28 December 2021
Accepted: 10 February 2022
Published: 25 March 2022
Issue date: April 2022

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We thank research funding from National Natural Science Foundation of China (21874147).

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