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Evanescent-wave microscopy achieves sub-diffraction axial sectioning byconfining fluorescence excitation to a thin layer close to the cell/substrateinterface. How thin this light sheet exactly is, however, is often unknown.Particularly in the popular objective-type total internal reflectionfluorescence microscopy (TIRFM) configuration large deviations from theexpected exponential intensity decay of the evanescent wave have been reported.Propagating, i.e., non-evanescent, excitation light diminishes the opticalsectioning effect, reduces contrast and renders the quantification of TIRFMimages uncertain. Here, we use a combination of azimuthal- and polar-angle beamscanning, dark-field scatter imaging, and atomic force microscopy to identifythe sources of this unwanted background fluorescence excitation. We identifystray light originating from the microscope optics and the objective lensitself as the major sources of background, with minor contributions due toevanescent-wave scattering at the reflecting interface and at refractive-indexboundaries in the sample. Apart from evanescence in excitation light, lightemitted from a fluorophore can also show observable effects of evanescence.Only fluorophores located close to the coverslip can couple their near-fieldradiation into propagating waves detectable at supercritical angles. We showthat selectively detecting this supercritical-angle fluorescence (SAF) througha high-numerical aperture objective effectively rejects fluorescence fromdeeper sample regions and improves optical sectioning. The microscopy schemepresented here merges the benefits of TIRF excitation and SAF detection andprovides the conditions for quantitative wide-field imaging of fluorophoredynamics at or near the plasma membrane.

Combined evanescent-wave excitation and supercritical-angle fluorescence detection improves optical sectioning