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Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence.

Publication ,  Journal Article
Reichert, WM; Truskey, GA
Published in: Journal of cell science
June 1990

Total Internal Reflection Fluorescence (TIRF) is a powerful technique for visualizing focal and close contacts between the cell and the surface. Practical application of TIRF has been hampered by the lack of straightforward methods to calculate separation distances. The characteristic matrix theory of thin dielectric films was used to develop simple exponential approximations for the fluorescence excited in the cell-substratum contact region during a TIRF experiment. Two types of fluorescence were examined: fluorescently labeled cell membranes, and a fluorescent water-soluble dye. By neglecting the refractive index of the cell membrane, the fluorescence excited in the cell membrane was modelled by a single exponential function while the fluorescence in the membrane/substratum water gap followed a weighted sum of two exponentials. The error associated with neglecting the cell membrane for an incident angle of 70 degrees never exceeded 2.5%, regardless of the cell-substratum separation distance. Comparisons of approximated fluorescence intensities to more exact solutions of the fluorescence integrals for the three-phase model indicated that the approximations are accurate to about 1% for membrane/substratum gap thicknesses of less than 50 nm if the cytoplasmic and water gap refractive indices are known. The intrinsic error of this model in the determination of membrane/substratum separations was 10% as long as the uncertainties in the water gap and cytoplasmic refractive indices were less than 1%.

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Published In

Journal of cell science

DOI

EISSN

1477-9137

ISSN

0021-9533

Publication Date

June 1990

Volume

96 ( Pt 2)

Start / End Page

219 / 230

Related Subject Headings

  • Water
  • Models, Biological
  • Microscopy, Fluorescence
  • Mathematics
  • Glass
  • Fluorescence
  • Developmental Biology
  • Cell Membrane
  • Cell Adhesion
  • 3101 Biochemistry and cell biology
 

Citation

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Reichert, W. M., & Truskey, G. A. (1990). Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence. Journal of Cell Science, 96 ( Pt 2), 219–230. https://doi.org/10.1242/jcs.96.2.219
Reichert, W. M., and G. A. Truskey. “Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence.Journal of Cell Science 96 ( Pt 2) (June 1990): 219–30. https://doi.org/10.1242/jcs.96.2.219.
Reichert WM, Truskey GA. Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence. Journal of cell science. 1990 Jun;96 ( Pt 2):219–30.
Reichert, W. M., and G. A. Truskey. “Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence.Journal of Cell Science, vol. 96 ( Pt 2), June 1990, pp. 219–30. Epmc, doi:10.1242/jcs.96.2.219.
Reichert WM, Truskey GA. Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence. Journal of cell science. 1990 Jun;96 ( Pt 2):219–230.
Journal cover image

Published In

Journal of cell science

DOI

EISSN

1477-9137

ISSN

0021-9533

Publication Date

June 1990

Volume

96 ( Pt 2)

Start / End Page

219 / 230

Related Subject Headings

  • Water
  • Models, Biological
  • Microscopy, Fluorescence
  • Mathematics
  • Glass
  • Fluorescence
  • Developmental Biology
  • Cell Membrane
  • Cell Adhesion
  • 3101 Biochemistry and cell biology