Characterization and improvement of highly inclined optical sheet microscopy

Highly Inclined and Laminated Optical sheet (HILO) microscopy is an optical technique that employs a highly inclined laser beam to illuminate the sample with a thin sheet of light that can be scanned through the sample volume. HILO is an efficient illumination technique when applied to fluorescence imaging of thick samples owing to the confined illumination volume that allows high contrast imaging while retaining deep scanning capability in a wide-field configuration. The restricted illumination volume is crucial to limit background fluorescence originating from portions of the sample far from the focal plane, especially in applications such as single molecule localization and super-resolution imaging. 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 optimal application to single molecule and super-resolution imaging. We demonstrate that, by reducing the beam size along the refracted axis only, the excitation volume is consequently reduced while maintaining a field of view suitable for single cell imaging. We quantify the enhancement in signal-tobackground ratio with respect to the standard HILO technique and apply our illumination method to dSTORM superresolution imaging of the actin and vimentin cytoskeleton. We define the conditions to achieve localization precisions comparable to state-of-the-art reports, obtain a significant improvement in the image contrast, and enhanced plane selectivity within the sample volume due to the further confinement of the inclined beam.