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Characterisation of biocondensate microfluidic flow using array-detector FCS.
Stijn Dilissen1, Pedro L Silva2, Anastasia Smolentseva2
1UHasselt, Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre, Biomedical Research Institute, Agoralaan C (BIOMED), B3590 Diepenbeek, Belgium; UHasselt, Biomedical Device Engineering group, Institute for Materials Research (IMO-IMOMEC), Wetenschapspark 1, B3590 Diepenbeek, Belgium.
Array detector fluorescence correlation spectroscopy (AD-FCS) enables single-molecule studies of Tau protein condensates under flow. This method quantifies flow velocity and condensate size distribution, advancing liquid-liquid phase separation research.
Area of Science:
- Biophysics
- Cell Biology
- Biochemistry
Background:
- Liquid-liquid phase separation (LLPS) drives cellular organization, but quantitative methods for studying biocondensates are limited.
- Biocondensate rheology and constituent dynamics are functionally critical but challenging to measure.
- Standard methods relying on Brownian diffusion are unsuitable for dense condensates that sediment.
Purpose of the Study:
- To develop and validate a single-molecule fluorescence technique for studying biomolecular condensates under flow.
- To investigate the properties of Tau protein condensates using microfluidics and advanced fluorescence correlation spectroscopy.
- To establish a method for characterizing condensate dynamics and size distribution.
Main Methods:
- Utilized a microfluidic chip for controlled flow experiments with Tau protein condensates.
- Employed array detector fluorescence correlation spectroscopy (AD-FCS) for enhanced molecular analysis.
- Combined microfluidics with AD-FCS to measure flow profiles, condensate flow, and burst durations.
Main Results:
- AD-FCS successfully characterized the 3D flow profiles within the microfluidic chip.
- Mapped the flow behavior of Tau condensates and measured their residence times.
- Obtained flow velocity and burst duration data, enabling estimation of condensate size distribution.
Conclusions:
- AD-FCS is a powerful tool for single-molecule studies of biocondensates under flow conditions.
- This technique offers a convenient method for estimating condensate size distribution.
- AD-FCS opens new avenues for investigating biocondensate phase diagrams and properties.
