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.

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