When investigating the interaction of cells with surfaces, it is becoming increasingly important to perform quantitative measurements of surface protein density to understand reaction kinetics. Previously, to calibrate a surface for an experiment one would have to use a radiometric assay or strip the surface with acid and perform a mass quantification. Although both of these methodologies have been proven to be effective measurement techniques for surface quantification, they can be time consuming and require substantial amounts of material. The latter is particularly problematic when working with specialized molecules or constructs that may be expensive to produce and/or only available in small quantities. Here we present a simple method to measure the intensity and penetration depth of an evanescent wave, and use this information to quantify the density of surface molecules in a microscopic region of a transparent surface.
Keywords: Fluorescence calibration; surface coating; surface concentration; total internal reflectance.
© 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.