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Biomolecules
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Studies of Protein Dynamics, Interactions, and Concentration by Fluorescence Fluctuation...
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Studies of Protein Dynamics, Interactions, and Concentration by Fluorescence Fluctuation Correlation Spectroscopy (FFCS)

A special issue of Biomolecules (ISSN 2218-273X).

Deadline for manuscript submissions: 31 December 2026 | Viewed by 3111

Special Issue Editors

Dr. Jieqiong Lou

E-Mail Website
Guest Editor
School of Biomedical Sciences, University of Melbourne, Melbourne, Australia
Interests: chromatin organization; light microscopy; biochemistry and cell biology; medical biochemistry and metabolomics; fluorescence microscopy; cell biology; protein dynamics; membrane protein signaling and trafficking
Special Issues, Collections and Topics in MDPI journals
Dr. Elvis Pandžić

E-Mail Website
Guest Editor
Katharina Gaus Light Microscopy Facility, Mark Wainwright Analytical Centre, Lowy Cancer Research Centre C25, University of New South Wales, Sydney, NSW 2052, Australia
Interests: fluorescence fluctuation correlation spectroscopy; membrane biophysics; mechanobiology; super-resolution microscopy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues, 

Biomolecules will publish a Special Issue, entitled “Studies of Protein Dynamics, Interactions, and Concentration by Fluorescence Fluctuation Correlation Spectroscopy (FFCS)”, highlighting the power and versatility of FFCS techniques in terms of exploring molecular dynamics in live cells and complex biological environments. This Issue aims to showcase advances in both the methodology and application of FFCS, including but not limited to fluorescence correlation spectroscopy (FCS), image correlation spectroscopy (ICS), pair correlation function analysis, line-scan confocal fluctuation analysis, and related extensions.

We welcome contributions that explore the development of new imaging protocols, experimental designs, and analysis tools, as well as those applying FFCS-based approaches to reveal critical insights into biomolecular behavior in living systems. 

Topics of interest: 

  • The evaluation of commercial and custom-built microscope systems for FFCS and related techniques.
  • The development of novel acquisition strategies, analysis software, and plugins for FFCS-based methods.
  • The applications of FFCS techniques to study protein diffusion, flow, confinement, kinetics, interactions, and concentration heterogeneity in biological systems. 

This Special Issue is intended to serve both the biophysics and cell biology communities, and we invite scholars to contribute high-quality, relevant submissions. Please contact the Guest Editors if you have any questions regarding scope or suitability. 

We are pleased to announce that the following leading experts in the field will be contributing to this Special Issue: 

Prof. Wiseman—"Measurement of membrane proteins transport and confinement by lipidic and protein complexes using k-space Image Correlation Spectroscopy";

Prof. Clayton—"Polarized phase-sensitive fluorescence image correlation microscopy";

Prof. Wohland—"ImFCS study of membrane-associated Myosin Light Chain in focal adhesion formation";

Prof. Maiti—"TBC";

A. Prof. Lanzanò — "Correction of background in Fluorescence Correlation Spectroscopy for the accurate determination of particle number";

A. Prof. Hinde—"Using Fluorescence Correlation Spectroscopy to Study Protein Dynamics from the Plasma Membrane to the Nucleus".

Dr. Jieqiong Lou
Dr. Elvis Pandžić
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 250 words) can be sent to the Editorial Office for assessment.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Biomolecules is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • fluorescence fluctuation correlation spectroscopy (FFCS)
  • fluorescence correlation spectroscopy (FCS)
  • image correlation spectroscopy (ICS)
  • pair correlation function (pCF) analysis
  • line-scan correlation
  • protein dynamics
  • molecular diffusion
  • molecular flow
  • confinement dynamics
  • protein–protein interactions
  • membrane organization
  • intracellular transport
  • molecular crowding
  • spatiotemporal fluctuations
  • concentration heterogeneity
  • live-cell imaging
  • single-molecule analysis
  • quantitative fluorescence microscopy
  • biophysics
  • super-resolution fluctuation analysis

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Further information on MDPI's Special Issue policies can be found here.

Published Papers (4 papers)

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Research

17 pages, 6733 KB  
Article
Ghosts on the Membrane: Cytoskeletal Pinning Influences Nanoscale Cell Membrane Organization
by Shambhavi Pandey and Thorsten Wohland
Biomolecules 2026, 16(4), 596; https://doi.org/10.3390/biom16040596 - 17 Apr 2026
Viewed by 449
Abstract
The lateral organization of the plasma membrane (PM) is vital for cellular signaling, yet the specific mechanisms by which the internal cortical actin meshwork templates the organization of the external lipid leaflet remain poorly understood. While established models like the ‘picket-fence’ emphasize physical [...] Read more.
The lateral organization of the plasma membrane (PM) is vital for cellular signaling, yet the specific mechanisms by which the internal cortical actin meshwork templates the organization of the external lipid leaflet remain poorly understood. While established models like the ‘picket-fence’ emphasize physical barriers to diffusion, recent observations of fiber-like “ghost” structures in the distribution of glycosylphosphatidylinositol-anchored proteins (GPI-APs) suggest a more intricate mode of spatial coordination. In this study, we utilize imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) and variable-angle TIRF to resolve whether these filamentous patterns represent genuine membrane-proximal features or optical artifacts of cytosolic transport. Our results demonstrate that these fiber-like tracks are strictly confined to the immediate PM interface and disappear as the evanescent field probes deeper into the cytosol. While the spatial distribution of GPI-APs is templated by the underlying actin meshwork, quantitative diffusion mapping shows that the lateral dynamics of the probe remains largely uniform and is not significantly modulated by these filamentous patterns. By pharmacologically perturbing the actin scaffold and membrane cholesterol, we show that this transbilayer coupling is contingent upon a cholesterol-dependent cytoskeletal pinning mechanism. These findings demonstrate a decoupling of spatial organization and molecular dynamics, providing evidence for how the actin scaffold patterns nanoscale membrane organization without imposing long-range barriers to diffusion. Full article
(This article belongs to the Special Issue Studies of Protein Dynamics, Interactions, and Concentration by Fluorescence Fluctuation Correlation Spectroscopy (FFCS))
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23 pages, 2217 KB  
Article
Measurement of Protein Transport in Heterogeneous Environments Using Confinement k-Space Image Correlation Spectroscopy
by Elvis Pandžić, John W. Hanrahan, Asmahan Abu-Arish and Paul W. Wiseman
Biomolecules 2026, 16(4), 519; https://doi.org/10.3390/biom16040519 - 31 Mar 2026
Viewed by 444
Abstract
Current models of the cell membrane assume a heterogenous environment such as sphingomyelin and cholesterol-enriched nano- and microdomains, which are thought to functionally sequester proteins. Besides lipid-ordered domains, membrane proteins can interact with protein complexes by transient binding—necessary for their functional role. Here, [...] Read more.
Current models of the cell membrane assume a heterogenous environment such as sphingomyelin and cholesterol-enriched nano- and microdomains, which are thought to functionally sequester proteins. Besides lipid-ordered domains, membrane proteins can interact with protein complexes by transient binding—necessary for their functional role. Here, we show that an extension of k-space Image Correlation Spectroscopy applied to standard fluorescence microscopy image time series can be used to characterize the protein confinement in heterogeneous membranes. To validate this method, we simulated confined diffusion of tracer particles in a system of static microdomains where we varied the domain size, domain density, confinement probability and diffusion coefficients of tracer particles. We show how the kICS correlation function changes with these parameters and gives rise to emergent properties of the system such as apparent domain sizes and characteristic diffusion coefficients. As a validity check, we apply this analysis to study the dynamics of lipid domain-associated glycosylphosphatidyl inositol (GPI)-anchored proteins labeled by green fluorescent proteins (GPI-GFP) in intact COS-7 cell membranes, and upon domain-disrupting enzyme treatments. Full article
(This article belongs to the Special Issue Studies of Protein Dynamics, Interactions, and Concentration by Fluorescence Fluctuation Correlation Spectroscopy (FFCS))
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18 pages, 2157 KB  
Article
Polarized Phase-Sensitive Fluorescence-Image Correlation Spectroscopy
by Andrew H. A. Clayton
Biomolecules 2026, 16(3), 433; https://doi.org/10.3390/biom16030433 - 13 Mar 2026
Viewed by 485
Abstract
Molecular interactions underpin the functioning of the living cell. Molecules exist in distinct quaternary structural forms, associate with molecular partners in signaling cascades, form transient quinary interactions, localize in membrane domains, and cluster in membrane-less condensates. Measuring the concentration, size, and dynamics of [...] Read more.
Molecular interactions underpin the functioning of the living cell. Molecules exist in distinct quaternary structural forms, associate with molecular partners in signaling cascades, form transient quinary interactions, localize in membrane domains, and cluster in membrane-less condensates. Measuring the concentration, size, and dynamics of these molecular assemblies remains an enduring biophysical challenge, particularly in cells, where heterogeneity is the rule rather than the exception. Orthogonal signals derived from fluorescence lifetime, fluorescence fluctuations, and fluorescence polarization provide valuable metrics for probing interactions and environments, concentration and size, and rotational dynamics, respectively. This paper combines fluorescence lifetime imaging microscopy with image correlation analysis and polarization to determine the concentrations, brightness, lifetime, and rotational correlation time of different fluorescent states. A two-population model is examined as a prototypical example of a heterogeneous system. The analysis is illustrated on a simple fluorescence model system, where cluster densities, relative brightnesses, lifetimes, and rotational correlation times are extracted. Full article
(This article belongs to the Special Issue Studies of Protein Dynamics, Interactions, and Concentration by Fluorescence Fluctuation Correlation Spectroscopy (FFCS))
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14 pages, 3679 KB  
Article
Correction of Background in Fluorescence Correlation Spectroscopy for Accurate Determination of Particle Number
by Elisa Longo, Greta Paternò, Elisabetta Di Franco, Paolo Bianchini, Marco Castello, Alberto Diaspro, Giuseppe Vicidomini, Elena Bruno, Paolo Musumeci, Maria Josè Lo Faro, Nunzio Tuccitto and Luca Lanzanò
Biomolecules 2026, 16(1), 11; https://doi.org/10.3390/biom16010011 - 20 Dec 2025
Viewed by 1154
Abstract
Since the early development of Fluorescence Correlation Spectroscopy (FCS), it has been recognized that background intensity can lead to artifacts in the amplitude of the autocorrelation function (ACF) and, consequently, to inaccurate estimates of particle numbers. Here, we present a protocol for quantitative [...] Read more.
Since the early development of Fluorescence Correlation Spectroscopy (FCS), it has been recognized that background intensity can lead to artifacts in the amplitude of the autocorrelation function (ACF) and, consequently, to inaccurate estimates of particle numbers. Here, we present a protocol for quantitative background evaluation and amplitude correction in FCS experiments, applicable to different sources of background such as detector noise, autofluorescence, and light scattering. We demonstrate the performance of our approach through three representative case studies: (i) FCS measurements of a bright fluorophore at low concentration, (ii) FCS of dim nanoparticles affected by solvent Raman scattering, and (iii) FCS performed using a confocal setup equipped with a SPAD array, where background originates from detector hot pixels. These examples represent typical experimental conditions in which background signals compromise quantitative interpretation, illustrating how our protocol restores accuracy and reproducibility in FCS analysis. By systematically identifying and correcting these effects, the proposed protocol addresses a long-standing limitation of FCS and provides a robust framework for improving the accuracy and reproducibility of quantitative fluorescence measurements. Full article
(This article belongs to the Special Issue Studies of Protein Dynamics, Interactions, and Concentration by Fluorescence Fluctuation Correlation Spectroscopy (FFCS))
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