Principle of fluorescence correlation spectroscopy and the basic workflow of imaging FCS — Principle of FCS and the basic workflow of imaging FCS. Krieger et al., *Nature Protocols* (2015).

Biophysical Fluorescence Laboratory

Deciphering dynamics at different biological length and time scales by fluorescence

Research

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Dual-colour imaging of F-tractin and PMT-mEGFP-F-tractin in live cells, with diffusion coefficient distributions on and off actin fibres

Imaging FCS combined with dual-colour imaging lets us see how actin fibres shape membrane dynamics in live cells. A membrane-anchored probe that binds actin diffuses markedly slower on fibres than between them, revealing how the cytoskeleton organises the plasma membrane.

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Z-scanning light-sheet FCS measuring diffusion inside and outside a bacterial biofilm

Bacteria and biofilms

Z-scanning light-sheet imaging FCS maps how fluorescent molecules such as dextrans and antibiotics diffuse in and around bacterial biofilms. Diffusion coefficient maps show molecules slowing sharply once they enter the biofilm, revealing how its structure limits penetration.

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Temperature- and divalent-cation-dependent expansion and contraction of the DENV2 envelope, measured by FRET

Viruses

We observe hitherto unseen conformational changes and structural dynamics of the DENV2 envelope that are influenced by both temperature and divalent cations. Our results show that for DENV2 and DENV1 the intrinsic dynamics, but not the specific morphologies, are correlated with viral infectivity.

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Zebrafish

Using SPIM-based spatial fluorescence cross-correlation spectroscopy over 1 to 10 µm, we follow how the morphogen Squint moves through the extracellular space of live zebrafish embryos. Separating free diffusion from receptor binding shows that transport slows with distance in narrow gaps between cells but stays fast in wider spaces.

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Cost-effective dark-field scattering correlation spectroscopy setup, samples and analysis

Instrumentation

We developed a cost-effective dark-field scattering correlation spectroscopy platform for quantitative measurement of nanoparticle dynamics. Validated across GNPs in solution, supported lipid bilayers, and cell membranes, the platform enables detection of mobility changes and provides a foundation for antimicrobial screening based on membrane disruption.

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Bilayers

Using imaging total internal reflection FCS and molecular dynamics simulations on supported lipid bilayers, we showed how the plant dehydrin Lti30 protects membranes during cold and drought stress: it binds lipids electrostatically in a pH-dependent way, cross-linking them and restricting their mobility.

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Martini 3 coarse-grained molecular dynamics snapshots of a DIPC/DPPC/cholesterol bilayer phase separating from 0 ns to 20 microseconds

Simulations

Martini 3 coarse-grained MD snapshots (0 ns to 20 µs) of a DIPC/DPPC/Chol (4:4:3) bilayer undergoing spontaneous phase separation.

Architecture of the ImFCSNet and FCSNet deep-learning networks compared with nonlinear least-squares fitting, with resulting diffusion coefficient maps

AI and ML

The deep-learning networks FCSNet and ImFCSNet predict diffusion coefficients more precisely than conventional fitting while needing far less data, cutting a typical Imaging FCS measurement from about 100 MB to 5 MB and opening the way to real-time measurements.

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Resources

Free software and tutorials for anyone working with FCS.

Software

Imaging FCSImageJ / Fiji plugin FCS PlotterFit and plot curves Imaging FCSAPEER cloud module TRASTTransient state analysis Confocal FCSAnalysis software

External tools and tutorials

QuickFit 3On GitHub Bayes FCSModel selection Confocal FCSInteractive tutorial Imaging FCSInteractive tutorial FCS tutorialStart here