Quantifying F-actin patches in single melanoma cells using total-internal reflection fluorescence microscopy

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Total-internal reflection fluorescence (TIRF) microscopy selectively excites fluorophores near a cell's contact area.
• Quantitative analysis using TIRF is challenging due to excitation intensity variations with axial position.
• Existing methods struggle to provide precise measurements of cellular components.

Purpose of the Study:

• To develop an easy-to-implement data analysis method for quantitative characterization of fluorescent signals in TIRF microscopy.
• To establish new parameters for analyzing F-actin patches in melanoma cells at the cell-substrate interface.
• To enable quantitative insights into cell dynamics and F-actin organization.

Main Methods:

• Developed a novel data analysis approach for TIRF microscopy images, focusing on signal characteristics rather than intensity values.
• Defined and applied two key quantitative parameters: elongation and surface density of F-actin patches.
• Utilized F-actin patches in single melanoma cells as a model system for validation.

Main Results:

• Introduced 'elongation' parameter to assess F-actin patch dispersion and classify cell spreading stages.
• Introduced 'surface density profile' to probe spatio-temporal distribution of F-actin patches.
• Demonstrated the method's effectiveness in characterizing F-actin organization at the cell contact area.

Conclusions:

• The proposed data analysis method overcomes TIRF microscopy's quantitative limitations without intensity calibration.
• Elongation and surface density parameters provide valuable insights into cell dynamics and F-actin organization.
• The analysis techniques are broadly applicable to other advanced optical microscopy methods for image analysis.