Imaging optically thick tissues simply and reproducibly: a practical guide to Lightsheet Macroscopy

Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting glitchy and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardized 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes should be non-aberrating with objective numerical apertures less than 0.65, and present an inexpensive tool for alignment and calibration of standard lightsheet parameters. Mouse embryo, liver and heart imaging are demonstrated as examples with practical recommendations for acquisition and post-processing. ### Competing Interest Statement The authors have declared no competing interest.