Epi-illumination dark-field microscopy enables direct visualization of unlabeled small organisms with high spatial and temporal resolution

Dark-field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under-explored and so far, only meso-scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans- and epi-illumination configurations. Our findings suggest that the epi-illumination configuration is superior in both contrast and fidelity compared to trans-illumination, while having the added advantage of experimental simplicity and an “open top” for experimental intervention. Guided by the theoretical analysis, we constructed an epi-illumination dark-field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans.     

A Method for Thin Sectioning Heavily Sclerotized Cuticle of Odontotaenius disjunctus

Elytra of the bessbeetle, Odontotaenius disjunctus were thin sectioned after embedding in epoxy resin. Sections were cut with a diamond saw, ground to the desired thickness on a rotary grinder and polished. Tearing and distortion were reduced when compared to knife-cut sections of heavily sclerotized cuticle.     

Macroscopic Sectioning of Undecalcified Tissues

An inexpensive laboratory apparatus designed to section undecalcified teeth and bone has been constructed. It consists of an electric motor with a mandrel bearing a carborundum sectioning disk centered within a Plexiglas enclosure. A coolant flows from a reservoir positioned in the upper portion of the Plexiglas enclosure to prevent desiccation or burning of the tissues. Undecalcified tissue can be cut into a series of thin, 0.5-1 mm slices to facilitate studies of pulpal enzymes, tooth morphology and design of dental cavity preparations.     

Chemical sectioning fluorescence tomography: high-throughput,high-contrast,multicolor, whole-brain imaging at subcellular resolution

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