Super-resolution measurements with evanescent-wave fluorescence-excitation using variable beam incidence

摘要: The evanescent wave (EW) elicited by total internal reflection of light selectively excites fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present epiillumination microscopy, and--close to the coverslip--can offer fivefold enhancement axial sectioning compared confocal and two-photon microscopy. decay length field is function refractive indices wavelength involved, modulated beam angle. microscopy was used study distribution concentration at or near interface presence high concentrations bulk solution. We modified upright microscope accommodate condenser optics needed for excitation. Systematic variations angle incidence were attained using acousto-optical deflector, telecentric optics, hemicylindrical prism. three-dimensional reconstruction fluorophore from angle-resolved image stacks results topographical information with resolution tens nanometers. applied this technique position dye-labeled subcellular storage organelles ('vesicles') approximately 300 nm diameter "footprint" region living neuroendocrine cells grown on