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Variable Incidence Angle Fluorescence Interference Contrast Microscopy for Z-Imaging Single Objects

Department of Chemistry, Stanford University, Stanford, California 94305-5080

Correspondence: Address reprint requests to Steven G. Boxer, Tel.: 650-723-4482; Fax: (650) 723-4817; E-mail: sboxer{at}stanford.edu.

Surface-generated structured illumination microscopies interrogate the position of fluorescently labeled objects near surfaces with nanometer resolution along the z axis. However, these techniques are either experimentally cumbersome or applicable to a limited set of experimental systems. We present a new type of surface-generated structured illumination fluorescence microscopy, variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC), in which the fluorescent sample is assembled above a reflective Si surface and the incidence angle of excitation light is varied by placing annular photomasks with different radii in the aperture diaphragm plane of the microscope. The variation in incidence angle alters the interference pattern of excitation light, and hence the intensity of detected fluorescence. Quantitative VIA-FLIC is tested by using a set of fluorophore-containing supported membranes separated from the Si surface by SiO₂ layers of variable thicknesses. The resulting fluorescence intensity versus incidence angle curves depends on the separation from the Si surface and when fit with an appropriate model yield precise SiO₂ thicknesses that are accurate with respect to the known SiO₂ thicknesses. Since only a simple modification to a standard epifluorescence microscope is required, VIA-FLIC offers a versatile method to produce z-reconstructions with high resolution for a wide range of biological systems.

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