Quantifying spatial and temporal variations of the cell membrane ultra-structure by bimFCS

Area of Science:

• Cell Biology
• Biophysics
• Membrane Dynamics

Background:

• Cell membranes exhibit heterogeneity across various scales, influencing receptor diffusion and cell processes.
• Understanding lipid-protein organization and cell cortex interactions is crucial for cell signaling.
• Existing methods like single particle tracking and FCS offer insights into component motion.

Purpose of the Study:

• To review a combined Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS) method for analyzing cell membrane organization.
• To demonstrate the simultaneous interrogation of all length scales in membrane diffusion.
• To quantify membrane nanodomains and cell cortex interactions in living cells.

Main Methods:

• Utilizes a combination of Fluorescence Correlation Spectroscopy (FCS) and Total Internal Reflection Microscopy (TIRF).
• Employs a spatially resolved detector, such as a camera with a 2D extended excitation profile.
• Requires minimal data acquisition time (seconds) for analysis.

Main Results:

• The combined TIR-FCS method enables simultaneous quantification of membrane nanodomains and cortex-induced diffusion changes.
• It allows for rapid assessment of membrane structure alterations in response to perturbations.
• The choice of fluorescent probe dictates the detected membrane heterogeneity.

Conclusions:

• TIR-FCS is a powerful technique for resolving cell membrane organization and dynamics in living cells.
• This method provides insights into complex cell physiology by analyzing spatial and temporal membrane structure changes.
• The review covers instrumentation, sample preparation, experimental, and computational needs, along with limitations.