WT and Tg mice are treated experimentally. Perfused and fixed brains are cleared, stained and imaged in the Mesoscale Imaging System.

AI-powered workflows in our 3TK software identify individual cells/objects throughout the brain and extract intensity, size and shape metrics.

Morphing experimental brains onto the Allen Reference Brain allows for statistical anatomic evaluation of object level features across 100’s of brain regions.

Time lapse video of the optical clearing step in Translucence’s tissue staining and clearing protocol. This brain is held in place with one of the tissue clamps that comes with the Mesoscale Imaging System.

Clearing and staining of a whole brain from a 5xFAD Alzheimer’s Disease model mouse with an antibody recognizing β-Amyloid.

AI-powered tools determine the probability that an object is a plaque (red) or cell (green). Our 3TK workflows can then perform brain-wide regional quantification of independent object populations extracted from the same fluorescence channel.

Regional quantification of Npas4 throughout the brain in a mouse left in the dark and exposed to light for various amounts of time. Our 3TK software applies a series of deterministic filters to the AI-powered probability maps to identify individual cells. Experimental brains are morphed onto the Allen Reference Brain and Npas4 expression levels are measured across 100’s of brain regions and analyzed without statistical anatomics methods.

Traditional immediate-early genes (IEG’s) have served as great markers of neuronal activity, however they are also regulated via neuromodulators via cAMP and by other paracrine factors.

Npas4 is exquisitely tuned to Ca²+ signaling downstream of neuronal activity providing a purer readout of electrogenic signals.

Our methods allow us to perform whole-brain immunostaining for both cFos and Npas4, identifying overlapping, yet distinct, populations of neurons.

These images are from the visual cortex after light exposure (left) and 3TK-identified cell labels (right). These images are from similar, but not overlapping fields. In overlapping fields, there are marked differences in the cFos(+) and Npas4(+) populations.

These 2D heatmaps are drawn from 3D whole-brain data sets and show the level of Npas4 expression determined from the Npas4 staining intensity of individual neurons.

The Npas4 signal is quantified in 100’s of brain regions over time. We have plotted data from a few exemplar regions in the bottom left.

The volcano plot shows brain regions in blue with statistically significant elevations of Npas4 expression after light exposure.

Exemplar optical slices from whole-brain immunostaining of microglia with an anti-Iba1 antibody. Brighter and more condensed microglia indicate there is activation throughout the brain when mice are treated with the inflammation-inducing agent, LPS.

The top images are a single z-plane through an intact 3D experimentally-treated brain. Iba1 immunoreactivity is shown on the left and the corresponding cell labels from a AI-powered workflow in our 3TK software are on the right.

Zoomed images show cellular morphology masks. Small seeming labels can be portions of microglia that project orthogonal to the 2D slice shown. Cell masks allow for counts, shape analysis and fluorescence intensity measurements.

The images on the left show optical slices through a control brain and a brain treated with the murine coronavirus, MHV (murine hepatitis virus). A large neuroinflammatory response is induced by viral treatment, with clear regional patterns of microglial activation.