Intern Spotlight on Nathan Hyman: No tissue clearing experience? No problem

As an emerging histological technique, tissue clearing is a powerful tool that allows scientists to image and analyze large intact samples. This new way of processing tissues allows researchers to uncover more comprehensive insights when compared to traditional 2D histology methods. By rendering whole samples transparent, tissue clearing techniques are able to maintain the three-dimensional structure of samples while deep tissue imaging via the use of a light sheet microscope.

While both scientifically relevant and powerful, widespread use of tissue clearing has been slow to adopt. Many scientists looking to integrate tissue clearing into their research often encounter many challenges like optimizing protocols and quantifying terabyte-scale datasets.

To empower scientists to adopt these techniques, Translucence Biosystems, with support from the National Institutes of Health (NIH), has developed user-friendly tissue clearing kits and AI-powered quantification software, Voxels. These kits deliver industry-quality clearing and staining directly to labs, without the need for specialized equipment or prior expertise.

To put these tools to the test, we gave them to our Undergraduate Intern, Nathan Hyman. Hyman joined Translucence Biosystems as a rising sophomore Undergraduate at the University of Michigan with one lab course under his belt. During the span of his internship, Hyman was able to generate 3D whole-brain anatomics data– with no prior lab experience and minimal hands-on help that would later be featured at the 2024 Society for Neuroscience meeting.

The following article describes how Hyman leveraged iDISCO-based tissue clearing methods, light sheet imaging, and Al-powered whole-brain object segmentation, to generate regionalized data across hundreds of brain regions.

Reflecting on his experience, Hyman shared, "The materials provided by Translucence outlined the protocol clearly, which gave me the confidence to approach these methods for the first time." By employing iDISCO-based tissue clearing, light sheet microscopy, and Voxels, Hyman was able to generate regionalized data for four distinct protein markers across five mouse brain specimens.

With the help of a Translucence Biosystems Tissue Clearing Kit, Hyman was able to clear whole mouse brains and stain a wide variety of epitopes, Iba1, Npas4, cFos, and tyrosine hydroxylase (TH), with no prior tissue clearing experience.

Nathan then used the Mesoscale Imaging System™, which adapts to the ZEISS Lightsheet Z.1 and 7 microscopes for imaging large intact tissues in high refractive index solutions with mesoscale optics, to image each of his samples in around an hour.

Nathan generated TIFF stacks of his samples using Stitchy™, a light sheet stitching tool delivering 2.5–5.5x faster performance than industry-leading commercial software solutions. Hyman was able to upload his raw CZI files and autonomously align them, in a matter of minutes, using Stitchy's auto-align feature. Hyman noted, "Stitchy™ was easy to use and was able to run on [his] laptop, which made things simple."

Following clearing, imaging, and stitching, Hyman was able to view his samples in 3D. "The nature of this data allowed me to see the whole brain at once and get a sense of epitope distribution across the whole brain." The image above is a max projection of tyrosine hydroxylase (TH) in one of the samples Hyman processed.

Above are images of two other epitopes that Hyman stained and imaged, Npas4 and cFos. Npas4 and cFos are immediate early genes (lEGs) that are expressed in response to recent neuronal activity. However, their expression patterns are both distinct and overlapping, as seen in the composite overlay of the two channels. For more information about Npas4 and cFos, and their role in whole-brain measurement of recent changes in neuronal activity, visit our science page.

Once all of his raw data was collected, Nathan was able to generate AI-powered quantification workflows to segment Iba1 (+) microglia and measure integrated TH intensity across the entire brain using Voxels™. Within Voxels™ Nathan was able to use the user-friendly Al-powered workflow builder which required no prior coding experience.

Above is an example of one of the segmentation workflows Nathan was able to generate with no prior experience using Voxels™. Raw microglia staining (left) and an example of the segmentation (right) show a small FOV of segmented Iba1(+) microglia which were segmented across whole brain samples.

Tissue clearing and imaging of Hyman's Tyrosine Hydroxylase (TH) labeled brain sample (left) shows fluorescence in key dopaminergic system areas including the visual cortex, substantia nigra, and striatum. The bar graph displays the integrated intensity of TH fluorescence across key brain regions, revealing expected high levels of expression in regions like the striatum and substantia nigra, while showing lower levels in regions typically associated with comparatively lower levels of TH expression like auditory areas.

Hyman's experience illustrates how Translucence Biosystems' tools empower researchers—even those without prior laboratory training—to generate high-quality 3D anatomic data. Translucence's tissue clearing kits enable industry-quality clearing and staining while their other tools support imaging, stitching, and the generation of AI-powered quantification workflows, for rapid and reliable analysis of intact tissues.

For more information on Hyman's successful application of 3D tissue clearing, 3D imaging, and 3D quantification to four distinct protein targets across five mouse brain samples, refer to his poster, "Lowering the Barrier to Tissue Clearing: Undergrad Approved", presented at the 2024 Society for Neuroscience meeting and the following news article.

Browse more of Hyman's work and related resources below.

‍

‍