Growing evidence suggests that certain psychiatric diseases such as schizophrenia and developmental disorders such as autism may involve subtle changes in specific neuronal cell-types. GABAergic interneurons that express the calcium-binding protein parvalbumin (PV) are one such cell-type, with PV-cells comprising about ~50% of all GABAergic interneurons and therefore being ~1 out of every 10 neurons in the brain. How does the number and distribution of PV-cells throughout the brains of mouse models of these disorders differ as a function of disease state? To address this question in the most unbiased manner, eFLASH whole hemisphere or whole brain labeling can be performed using LifeCanvas Technologies’ SmartLabel rapid immunohistochemistry device. Following SHIELD tissue preservation and optical clearing via delipidation in SmartClear II Pro, this intact mouse brain hemisphere was immunolabeled in only ~24 hours using just 20 µg of anti-PV antibody.

By refractive index matching the hemisphere and then imaging it in just ~30 minutes using LifeCanvas’s SmartSPIM light-sheet microscope, single-cell resolution data of the entire sample is acquired in one contiguous image volume, enabling holistic analysis of the tissue. Compared to cutting tissue into hundreds of thin sections and then imaging only those discrete regions of interest (ROIs) thought important to interrogate a specific hypothesis, unbiased study of the whole organ helps counteract spotlight biases and creates fertile ground where novel and unexpected discoveries can take place. By co-staining for additional cell-type markers and then analyzing the image data by aligning it to a reference atlas and quantifying number of PV-positive cells per brain region with automated cell-detection algorithms, a powerful pipeline to molecularly phenotype tissues can be created. With this approach, new insight into mouse models of these disorders could be obtained by investigating potential changes in the density and laminar & regional patterning of these cells, as well as changes in their co-expression of markers of neuronal activation (e.g., immediate early genes) or cell health (e.g., oxidative stress markers). Importantly, by performing these analyses and assessing the effects of candidate pharmacological interventions aimed at treating these conditions on a brain-wide basis, a much more complete picture than can be obtained from using only thin sections of tissue can be considered.

Xenograft models have been an essential research tool for neuro-oncology researchers. The establishment of orthotopic xenograft brain tumors requires the injection of tumor cells into relevant neuro-anatomical sites. Monitoring the spatial distribution and growth of intracranially-implanted tumors requires non-invasive live-imaging methods such as positron emission tomography (PET). Here, to assist researchers at the Barrow Neurological Institute validate a novel application of a PET tracer, LifeCanvas scientists preserved, cleared, labeled, and imaged at single-cell resolution mouse brain hemispheres that harbored xenografted striatal tumors. SYTO16 (cyan) was used as a nuclear stain and DyLight 649-conjugated tomato lectin (magenta) as a vascular stain, which together highlight the tumor’s morphology.

Had researchers at the Barrow Neurological Institute not tried LifeCanvas’s state-of-the-art tissue processing pipeline and instead used conventional histological methods it would have been a far more labor-intensive and time-consuming process, only to produce an image dataset that cannot be used as flexibly as the one generated by LifeCanvas. Specifically, our volumetric imaging capabilities enabled the researchers to easily and precisely visualize their orthotopic tumors in 3D and from whichever anatomical plane they desired, providing unparalleled insight into the extent of the tumor and its effects on the brain tissue.

Furthermore, our technologies can be broadly applied to tumor tissue from various models of cancer, including cell-line based and patient-derived xenografts. As cancer researchers increasingly shift towards more complex and pathologically-relevant model systems, it is critical that methods for extracting biological information can visualize and quantify molecular and structural information in 3D and at single-cell resolution. Our SHIELD tissue preservation strategies enable multiple rounds of immunolabeling, helping capture nuanced biological information that is only accessible from highly-multiplexed studies. For example, one could imagine using our tissue clearing and imaging pipeline to characterize local invasion and distant bone or brain metastases in animal models of breast cancer, staining for the requisite markers along the way. One could additionally assess how specific drug treatments simultaneously affect the immunological landscape and matrix composition of the tumor microenvironment, overlaying each new round of data in turn and building up a rich, holistic picture of the tissue.

LifeCanvas is the only company with the tools and capabilities necessary to achieve this unprecedented advance in tissue phenotyping, with our expertise available to customers both of our devices and of our Contract Research Services arm. By working with us, the possibilities are endless.

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.