Mirror-assisted light-sheet microscopy: a simple upgrade to enable bi-directional sample excitation

Light-sheet microscopy is a powerful imaging technique that achieves optical sectioning via selective illumination of individual sample planes. However, when the sample contains opaque or scattering tissues, the incident light sheet may not be able to uniformly excite the entire sample. For example, in the context of larval zebrafish whole-brain imaging, occlusion by the eyes prevents access to a significant portion of the brain during common implementations using unidirectional illumination. We introduce mirror-assisted light-sheet microscopy (mLSM), a simple inexpensive method that can be implemented on existing digitally scanned light-sheet setups by adding a single optical element-a mirrored micro-prism. The prism is placed near the sample to generate a second excitation path for accessing previously obstructed regions. Scanning the laser beam onto the mirror generates a second, reflected illumination path that circumvents the occlusion. Online tuning of the scanning and laser power waveforms enables near uniform sample excitation with dual illumination. mLSM produces cellular-resolution images of zebrafish brain regions inaccessible to unidirectional illumination. The imaging quality in regions illuminated by the direct or reflected sheet is adjustable by translating the excitation objective. The prism does not interfere with visually guided behavior. mLSM presents an easy-to-implement, cost-effective way to upgrade an existing light-sheet system to obtain more imaging data from a biological sample.