Confined diffusion in tubular structures analyzed by fluorescence correlation spectroscopy on a mirror

Area of Science:

• Biophysics
• Spectroscopy
• Cell Biology

Background:

• Standard fluorescence correlation spectroscopy (FCS) assumes free diffusion in large volumes, which is inaccurate in confined geometries like cellular tubules.
• This limitation leads to significant errors (up to 50%) in diffusion coefficient measurements in micrometer-range tubular structures.
• Such confined environments are common in living cells and microfluidic devices.

Purpose of the Study:

• To develop an improved FCS method for accurate molecular diffusion measurements in confined tubular structures.
• To overcome the limitations of standard FCS in geometries where the observation volume is comparable to the structure's dimensions.
• To validate the new method using fluorescent proteins in living bacterial cells.

Main Methods:

• Implementing FCS measurements in close proximity to a reflective surface (mirror).
• Utilizing the interference pattern (optical fringes) created by the excitation beam and its reflection.
• Analyzing the autocorrelation functions (ACFs) to extract diffusion information from fringe-confined diffusion.

Main Results:

• The proximity of a mirror introduces a new fluctuation time in the ACF, enabling accurate diffusion coefficient measurement.
• The method eliminates the need for assumptions about the confined geometry or the FCS observation volume.
• Accurate diffusion coefficients were measured for enhanced green fluorescent protein (EGFP) and IscS-EGFP in the cytoplasm of *Escherichia coli*.

Conclusions:

• Mirror-enhanced FCS significantly extends the validity range of measurements in tubular structures.
• This technique provides a robust way to measure real diffusion coefficients in confined cellular environments.
• The method is applicable to various fluorescent probes and cellular systems, including organelles and microfluidic devices.