<scp>CELS</scp>‐<scp>3D</scp>—Cutting edge light source for exciting fluorescence in microtome‐based <scp>3D</scp> microscopy and targeted correlative microscopy

We discovered that the light entering a triangular ultramicrotome glass knife from the bottom exits the knife through its cutting edge, forming an oblique light sheet illumination suitable for imaging. We adopted this light sheet for side illumination of the sample blocks during sectioning on the ultramicrotome, for 3D imaging, and for targeting fluorescent features for confocal-, electron- and correlative microscopy. In this paper, we present a working prototype named CELS-3D (Cutting Edge Light Source, Three-Dimensional), a microscope mounted on an ultramicrotome. We characterised CELS-3D and applied it for 3D imaging of human liver spheroids with a diameter of approximately 500 μm. The structure of nuclei and tight junctions has been successfully reconstructed over the full spheroid volume. In contrast, a confocal microscope was unable to image spheroids to a depth of greater than 50 μm. CELS-3D shows fluorescence during serial sectioning in an online mode; therefore, it can apply for targeting fluorescence structures for correlative microscopy. We successfully applied CELS-3D for targeted correlative microscopy of human liver spheroids and C. elegans. The CELS-3D can be utilised for less- and non-transparent samples, which encompasses a range of applications, including operation biopsies, experimental organoids/spheroids, artificial cartilage, and bone, among others. The CELS-3D can be effortlessly mounted on the top of any commercially available ultramicrotome, and its operation is straightforward and intuitive.