Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of...

Area of Science:

• Cell biology
• Biophysics
• Microscopy

Background:

• Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) enables controlled illumination depth.
• It has the potential to measure membrane/substrate separation distances and focal contact sizes.
• Bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye were used.

Purpose of the Study:

• To quantitatively determine absolute membrane/substrate separation distances.
• To measure individual focal contact areas using a simplified model of TIRFM optics.
• To assess the accuracy of VA-TIRFM for these measurements.

Main Methods:

• VA-TIRFM imaging of BAEC at varying illumination angles.
• Application of a simplified TIRFM optical model.
• Correction for background fluorescence and nonlinear regression analysis.
• Comparison with immunofluorescence staining of vinculin.

Main Results:

• Illumination of dorsal and ventral membranes occurred above 64 degrees.
• Focal contacts were selectively illuminated above 66 degrees.
• Background noise affected images above 74 degrees.
• Corrected analysis yielded focal contact separation distances of 24 +/- 13 nm.
• Focal contact areas measured by VA-TIRFM (1.3 +/- 0.7 micron2) closely matched vinculin staining results (1.5 +/- 0.3 microns2).

Conclusions:

• VA-TIRFM, with background correction, provides accurate measurements of focal contact separation distances and areas.
• The technique is valuable for studying cell-substrate interactions.
• Results align with established methods like vinculin staining.