High-resolution total-internal-reflection fluorescence microscopy using periodically nanostructured glass slides

We compare the performance of a total-internal-reflection fluorescence microscope under varying illumination and substrate conditions. The samples are deposited on a standard homogeneous glass slide or on a grating and illuminated by one or two interfering beams at various incident angles. A conjugate gradient with positivity a priori information is used to reconstruct the fluorophore density from the images. Numerical studies demonstrate that when the sample lies on an optimized grating, the lateral resolution of the microscope is greatly improved, up to fourfold, the best result being obtained when the grating is illuminated by two interfering beams. (C) 2009 Optical Society of America