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.
T. et al. (Mon,) studied this question.