Calibration Free FCS

Biomolecular condensates formed through liquid–liquid phase separation play a key role in organizing cellular processes by locally concentrating macromolecules without membrane boundaries. The measurement of molecular concentrations and mobilities within condensates remains challenging due to the heterogeneous nature of such systems, with conventional fluorescence correlation spectroscopy (FCS) techniques requiring external calibration, limiting their application in phase-separated systems.

To address this problem, Prerit Mathur and co-workers at ETH Zurich recently presented calibration-free scanning fluorescence correlation spectroscopy, a technique that enables direct, quantitative measurement of molecular concentrations and diffusivities without the need for external standards. Specifically, temporal line-scan FCS and sinusoidal scanning FCS are combined and used to measure diffusion coefficients and concentrations directly from scan-dependent fluorescence fluctuations. By decoupling concentration measurements from calibration assumptions, the approach allows consistent analysis across heterogeneous environments and over a wide range of concentrations.

To showcase the utility of this new method, the authors characterized phase separation of the intrinsically disordered region of the DEAD-box protein, Ddx4. Quantitative mapping of the phase diagram reveals a systematic decrease in molecular diffusivity within the dense phase as concentration increases. These results provide significant new insights into how phase separation modulates mobility and potentially stabilizes biomolecular condensates.

Written by Sabine Eisele

Read the published article here.