Fast submicrometer-scale imaging of whole zebrafish using the knife-edge scanning microscope

Advances in high-resolution 3D microscopy have enabled the investigation of subcellular microstructures in biological specimen. For a full understanding of the organism's structure and function, it is mandatory to obtain data from the whole animal, not just parts of it. In this paper, we present our work with the Knife-Edge Scanning Microscope (KESM) for imaging a Nissl-stained whole zebrafish larva. KESM combines a diamond microtome and line-scan imaging for simultaneous sectioning and imaging in 3D. We show that using the KESM, a zebrafish, less than 3 mm long and diameter less than 500 μm, can be imaged within 1 hour at a resolution of 0.6 μm × 0.7 μm × 1.0 μm. We also present new results on using a vibrating microtome to improve sectioning and imaging robustness.