Segmented fluorescence correlation spectroscopy (FCS) on a commercial laser scanning microscope.

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Accurate Fluorescence Correlation Spectroscopy (FCS) in cells is hindered by cellular dynamics.
• Segmented FCS analysis offers improved accuracy for intracellular measurements.
• Dedicated FCS modules are not always available on commercial microscopes.

Purpose of the Study:

• To demonstrate segmented FCS on a commercial laser scanning microscope (LSM) without a dedicated FCS module.
• To establish a method for acquiring and processing segmented FCS data using a custom MATLAB software.
• To measure molecular diffusion in specific subcellular regions with enhanced accuracy.

Main Methods:

• Data acquisition on a Leica SP8 confocal microscope.
• Export and processing of data using custom MATLAB software for segmented FCS analysis.
• Calculation of autocorrelation functions (ACF) and diffusion coefficients in defined subcellular areas.

Main Results:

• Validation of segmented FCS on a commercial LSM using fluorophores in solution.
• Accurate measurement of GFP diffusion in HeLa cell nucleoplasm and nucleolus, showing slower diffusion in the nucleolus.
• Determination of PARP1 chromobody diffusion, revealing slower mobility in the nucleolus compared to the nucleoplasm.

Conclusions:

• Segmented FCS is feasible on standard commercial laser scanning microscopes.
• The custom MATLAB software enables accurate intracellular diffusion measurements.
• This approach provides insights into molecular mobility within distinct subcellular compartments, such as the nucleolus and nucleoplasm.