Observing secretory granules with a multiangle evanescent wave microscope

In total internal reflection fluorescence microscopy (TIRFM), fluorophores near a surface can be excited with evanescent waves, which decay exponentia lly with distance from the interface. Penetration depths of evanescent wave s from 60 nm to 300 nm were generated by varying the angle of incidence of a laser beam. With a novel telecentric multiangle evanescent wave microscop e, we monitored and investigated both single secretory granules and pools o f granules in bovine chromaffin cells. By measuring the fluorescence intens ity as a function of penetration depth, it is possible through a Laplace tr ansform to obtain the fluorophore distribution as a function of axial posit ion. We discuss the extent to which it is possible to determine distances a nd diameters of granules with this microscopy technique by modeling the flu orescent volumes of spheres in evanescent fields. The anisotropic near-fiel d detection of fluorophores and the influence of the detection point-spread function are considered. The diameters of isolated granules between 70 nm and 300 nm have been reconstructed, which is clearly beyond the resolution limit of a confocal microscope. Furthermore, the paper demonstrates how eva nescent waves propagate along surfaces and scatter at objects with a higher refractive index. TIRFM will have a limited applicability for quantitative measurements when the parameters used to define evanescent waves are not o ptimally selected.