Perhaps more than any other area of the nervous system, applying tissue clearing and large format imaging techniques to the spinal cord offers researchers an unparalleled view into both its fine-scale cellular organization and broader topography. At over 4 cm long, more than 400 transverse histological sections, each 50 µm thick, would need to be cut to visualize just the proximal half of a mouse’s spinal cord. With a width of only ~2-3 mm these sections take skill to collect, handle, orient, and mount properly, with lost sections meaning lost information and an incomplete picture come imaging time. Upon acquisition of a complete set of whole-section images, registering them to one another to digitally reconstruct even a short length of the spinal cord can present a sizable challenge, with tracing of blood vessels, neuronal axons, and other radially-aligned structures of interest being even more difficult.
Enter modern tissue clearing, the approach of electrophoretically removing samples’ lipid-filled, light-scattering cell membranes and then rendering them optically transparent via incubation in aqueous solutions that raise and homogenize their refractive index. These cleared samples, such as the one provided by Prof. Helen Lai of UT Southwestern that is shown in the video, can then be imaged intact and without the need for sectioning, mounting, serial imaging, and registration. Thanks to an initial preservation step in which the sample was equilibrated in an epoxy-containing solution called SHIELD and then chemically cured to provide an extra layer of intermolecular crosslinking, fluorescence of the sample’s tdTomato molecules remained robust even after clearing. The bird’s-eye view that intact-sample approaches provide enable researchers to examine morphology without interruption, more easily detect the distribution & periodicity of rare cell-types and measure these properties accurately, and count & quantify each cell reliably as there is no ambiguity arising from cell bodies split across sections. Further, with LifeCanvas’s own SmartSPIM light-sheet microscope entire samples can be imaged rapidly, making it practical to avoid sampling-based stereological analysis methods that are tedious to employ and that have limitations in their applicability and interpretability.
To learn more about our light-sheet microscope SmartSPIM or to request a quote, click the button below or email email@example.com.
© Copyright LifeCanvas Technologies 2020. All Rights Reserved