Simple optical configuration for depth-resolved imaging using variable-angle evanescent-wave microscopy

摘要

Abstract: The evanescent wave (EW) elicited by total internal reflection of light provides a means to selectively excite fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epi-illumination microscopy, and can offer a 5-fold enhancement of axial resolution compared to confocal and two- photon microscopy. The decay length of the evanescent field is a function of the refractive indices at the interface, the wavelength of the light, and is modulated by the beam-angle. EW microscopy has been used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution on top of the interface. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. 3-D reconstruction of image stacks by an inverse Laplace transform results in topographical information with an axial resolution of 10's of nanometers. We have labeled subcelluar storage organelles ('vesicles') of approximately equal 300 nm diameter and visualized the trajectories of single vesicles in the 'footprint' region of living neuroendocrine cells, grown on the interface. !14