Before beginning your study, we will work with you to understand the strengths of our tissue-processing pipeline, how best to prepare your samples, what results to expect, and other key details critical to project success.
To process your samples, we first use a novel, polyepoxide-based fixation technique called SHIELD (Park et al, Nature Biotech, 2018) to maximally preserve the tissue’s endogenous biomolecules, including fluorescent proteins and targets for immunohistochemical labeling.
We then use detergents and electrophoresis (via SmartClear 2 Pro) to optically clear the tissue by removing lipid-filled cell membranes, which hinder antibody penetration and limit imaging depth by causing light to scatter.
Using SmartLabel, which incorporates our proprietary stochastic electrotransport technology (Kim et al, PNAS, 2015), we then label intact samples like mouse brain hemispheres with molecular probes such as IgG antibodies in just 1-3 days.
After clearing (and optional labeling), we turn translucent, delipidated samples fully transparent by incubating them in a solution (EasyIndex) that raises and homogenizes their refractive index, greatly enhancing penetration of the visible wavelengths of light. The sample is then mounted in an agarose gel for maximum stability during imaging, aiding colocalization of signals across channels.
Intact samples are then imaged in their entirety using our light-sheet microscope, SmartSPIM, generating datasets at a default voxel size (1.8 µm x 1.8 µm x 4 µm). Other acquisition parameters can be discussed in our initial consultation.
Using image volumes sufficient to identify each cell in 3D (e.g., using tissue injected with viral tracers, or following anti-cFos staining using SmartLabel), we can then quantify the labeling pattern in a number of ways to meet your needs.