Total Internal Reflection Fluorescence Measurements of Ion-Association Adsorption of Water-Soluble Porphyrins at Liquid/Liquid Interface

1995 ◽  
Vol 24 (4) ◽  
pp. 283-284 ◽  
Author(s):  
Hitoshi Watarai ◽  
Yoriko Saitoh
Keyword(s):  
Total Internal Reflection ◽  
Ion Association ◽  
Liquid Interface ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Water Soluble ◽  
Fluorescence Measurements ◽  
Adsorption Of Water

General electromagnetic theory of total internal reflection fluorescence: the quantitative basis for mapping cell-substratum topography

1987 ◽  
Vol 87 (5) ◽  
pp. 677-693
Author(s):  
D. Gingell ◽  
O.S. Heavens ◽  
J.S. Mellor
Keyword(s):  
Glass Surface ◽  
Total Internal Reflection ◽  
Initial Step ◽  
Electromagnetic Theory ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Water Soluble ◽  
Theoretical Predictions ◽  
Quantitative Basis ◽  
Aqueous Volume

Total internal reflection fluorescence (TIRF) has recently been used to look at the contacts made between cells and a glass surface on which they are spread. Our method utilizes the fluorescence of a water-soluble dye that acts as an extracellular aqueous volume marker. Fluorescence is stimulated by the short-range electric field near the glass surface that exists under conditions of total internal reflection. Since fluorescence is normally generated beneath a spread cell and not beyond it, the fluorescence of the image is related to the size of the cell-glass water gap. The images obtained are remarkable for their detail, contrast and the absence of confusing granularity due to cytoplasmic heterogeneity, which is commonly seen in interference reflection (IRM) images. We here develop a rigorous electromagnetic theory of total internal reflection in layered structures appropriate for cell contacts and apply it to quantitative TIRF. We show that: (1) TIRF, unlike IRM, can report cell-glass gaps in a way that is practically independent of the detailed physical properties of the cell; (2) TIRF is also far more sensitive than IRM for measuring cell-glass water gaps up to approximately equal to 100nm. These striking results explain the image quality seen by TIRF. As the initial step towards verifying our theory we show that measurement of the fluorescence stimulated by total internal reflection at a simple glass-water interface matches theoretical predictions.

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

1990 ◽  
Vol 96 (2) ◽  
pp. 219-230
Author(s):  
W.M. Reichert ◽  
G.A. Truskey
Keyword(s):  
Cell Membrane ◽  
Total Internal Reflection ◽  
Matrix Theory ◽  
Contact Region ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Water Soluble ◽  
Refractive Indices ◽  
Cell Contact ◽  
Characteristic Matrix

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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