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

Publication ,  Journal Article
Burmeister, JS; Truskey, GA; Reichert, WM
Published in: Journal of microscopy
January 1994

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).

Duke Scholars

Published In

Journal of microscopy

DOI

EISSN

1365-2818

ISSN

0022-2720

Publication Date

January 1994

Volume

173

Issue

Pt 1

Start / End Page

39 / 51

Related Subject Headings

  • Protein Binding
  • Microscopy, Fluorescence
  • Microscopy
  • Mathematics
  • Fibronectins
  • Endothelium, Vascular
  • Cells, Cultured
  • Cell Membrane
  • Cell Adhesion
  • Cattle
 

Citation

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ICMJE
MLA
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Burmeister, J. S., Truskey, G. A., & Reichert, W. M. (1994). Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts. Journal of Microscopy, 173(Pt 1), 39–51. https://doi.org/10.1111/j.1365-2818.1994.tb03426.x
Burmeister, J. S., G. A. Truskey, and W. M. Reichert. “Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts.Journal of Microscopy 173, no. Pt 1 (January 1994): 39–51. https://doi.org/10.1111/j.1365-2818.1994.tb03426.x.
Burmeister JS, Truskey GA, Reichert WM. Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts. Journal of microscopy. 1994 Jan;173(Pt 1):39–51.
Burmeister, J. S., et al. “Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts.Journal of Microscopy, vol. 173, no. Pt 1, Jan. 1994, pp. 39–51. Epmc, doi:10.1111/j.1365-2818.1994.tb03426.x.
Burmeister JS, Truskey GA, Reichert WM. Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts. Journal of microscopy. 1994 Jan;173(Pt 1):39–51.
Journal cover image

Published In

Journal of microscopy

DOI

EISSN

1365-2818

ISSN

0022-2720

Publication Date

January 1994

Volume

173

Issue

Pt 1

Start / End Page

39 / 51

Related Subject Headings

  • Protein Binding
  • Microscopy, Fluorescence
  • Microscopy
  • Mathematics
  • Fibronectins
  • Endothelium, Vascular
  • Cells, Cultured
  • Cell Membrane
  • Cell Adhesion
  • Cattle