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


Journal Article

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64 degrees, both the dorsal and ventral membranes were illuminated, while images excited above 66 degrees illuminated only focal contacts. Above 74 degrees the fluorescence of focal contacts was dominated by back-ground noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 +/- 13 nm. Focal contact areas estimated by TIRFM (1.3 +/- 0.7 micron2) agreed closely with areas observed by immunofluorescence staining of vinculin (1.5 +/- 0.3 microns2).

Full Text

Duke Authors

Cited Authors

  • Burmeister, JS; Truskey, GA; Reichert, WM

Published Date

  • January 1, 1994

Published In

Volume / Issue

  • 173 / Pt 1

Start / End Page

  • 39 - 51

PubMed ID

  • 8120882

Pubmed Central ID

  • 8120882

Electronic International Standard Serial Number (EISSN)

  • 1365-2818

International Standard Serial Number (ISSN)

  • 0022-2720

Digital Object Identifier (DOI)

  • 10.1111/j.1365-2818.1994.tb03426.x


  • eng