A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses

Area of Science:

• Biophysics
• Cellular Dynamics
• Advanced Microscopy Techniques

Background:

• Stimulated emission depletion-fluorescence correlation spectroscopy (STED-FCS) offers high spatio-temporal resolution for live-cell molecular dynamics.
• Incomplete fluorescence suppression in STED-FCS alters the point spread function (PSF), complicating data analysis.
• Accurate measurement of diffusion times and molecular numbers is crucial for understanding cellular processes.

Purpose of the Study:

• To develop and validate a new fitting model for STED-FCS data that accounts for incomplete fluorescence suppression.
• To improve the accuracy of measurements for molecular diffusion times and average molecule numbers.
• To demonstrate the applicability of the model using experimental STED-FCS data.

Main Methods:

• Theoretical modeling of the STED-FCS point spread function (PSF) considering incomplete fluorescence suppression.
• Development of a STED-background corrected fitting model for auto-correlation curves.
• Implementation of a STED module with a pulsed laser source on a commercial confocal/FCS microscope.
• Experimental validation of the model with varying STED power.

Main Results:

• The proposed model accurately describes the deviation of the STED-FCS PSF from a Gaussian shape.
• The STED-background corrected model significantly improves the accuracy of diffusion time and molecule number measurements.
• Experimental results show good agreement with theoretical predictions, with decreasing diffusion times and molecule numbers as STED power increases.

Conclusions:

• The new fitting model effectively addresses the challenges posed by incomplete fluorescence suppression in STED-FCS.
• This advancement enhances the reliability of nanoscale molecular dynamics studies in live cells.
• The validated model provides a more precise tool for quantitative analysis in advanced microscopy.