Characterization of highly inclined optical sheet microscopy for localization microscopy

Highly Inclined and Laminated Optical sheet (HILO) microscopy is an optical technique that employs a highly inclined laser beam to perform widefield fluorescence microscopy [1]. The confined illumination volume allows for better contrast with respect to standard epifluorescence microscopy, while retaining deep scanning capabilities. Limiting the background fluorescence originating from out-of-focus planes is especially crucial in applications such as single-molecule localization and super-resolution imaging [2,3]. Despite its widespread use, current literature lacks comprehensive reports of the actual advantages of HILO in these kinds of microscopies. Here, we thoroughly characterize the propagation of a highly inclined beam through fluorescently labeled samples and implement appropriate beam shaping for improving the performance of this technique in super-resolution imaging. We demonstrate that by reducing the beam size along the refracted axis only, we can further reduce the excitation volume and sensibly increase signal-to-background ratio of the images while still maintaining a field of view suitable for single-cell imaging. We then apply our illumination method to dSTORM super-resolution imaging of the vimentin cytoskeleton, and we demonstrate that the increased image contrast leads to better sampling of single molecules and ultimately to higher quality super-resolved images.