Our protocol would facilitate investigation of neuronal structures from circuit to component scales which is essential for better understanding of brain functions. Our clearing technique, ScaleSF exerts potent clearing capability, as well as a high level of tissue preservation that is required for simultaneous visualization of both large and small scale structures. Our protocol is especially effective in the brain where neuronal cells exhibit elaborate processes, tremendous links, and arrange specialized fine subcellular structures.
Before beginning the experiment, prepare Sca/eS solutions as shown in this table and cover the samples with foil. Start the procedure by adding eight milliliters each of ScaleS0 and ScaleS4 solutions to two separate wells of a six well cell culture plate and pre-warm the plate to 37 degrees Celsius in an incubator. Transfer the brain slices to the pre-warmed ScaleS0 solution with a spatula and incubate the slices for two hours at 37 degrees Celsius in a shaking incubator at 90 RPM.
Next, transfer the permeabilized brain slices in eight milliliters of PBS minus in a six well cell culture plate and wash for 15 minutes by keeping in an orbital shaker at 40 to 60 RPM. Repeat this step twice. Then, transfer the brain slices in eight milliliters of pre-warmed ScaleS4 solution and incubate for eight to 12 hours at 90 RPM at 37 degrees Celsius.
For chamber preparation, 3D print the chamber frame and microscope stage adapters. Attach the chamber frame to a coverslip using a pressure sensitive adhesive. Prepare 1.5%agarose in ScaleS4D25(0)solution.
Mix the solution and microwave it until the agarose is fully dissolved. Then allow the solution to cool to 37 degrees Celsius. Mount the cleared brain slice onto the bottom coverslip of the imaging chamber with a spatula.
Remove the excess solution from the cleared slice using a clean tissue. Add the ScaleS4 gel to the brain slice using a micropipette to fill the imaging chamber. Place another coverslip on top with forceps followed by a piece of tissue paper and a glass slide.
Transfer the imaging chamber to a refrigerator at four degrees Celsius. Place metal weights on the glass slide and leave them for 30 minutes. After the incubation, remove the material from the imaging chamber and wipe off excess gel.
Place the imaging chamber in a 60 millimeter glass Petri dish and attach the rim of the imaging chamber at multiple points to the dish with a pressure sensitive adhesive. Pour the ScaleS4 solution into the dish and shake gently for one hour at 40 to 60 RPM at 20 to 25 degrees Celsius. Substitute with fresh solution and remove air bubbles on the gel surface by gently scraping the surface using a pipette tip.
Mount the imaging chamber on a microscope stage. Prepare a confocal laser scanning microscope equipped with a multi-immersion objective lens of 2.5 millimeter working distance, 16x magnification and 0.60 numerical aperture. Turn on all the relevant imaging equipment, such as the workstation, microscope, scanner, lasers, mercury lamp, and launch an imaging software.
Set the correction collar of the multi-immersion objective lens to 1.47. Immerse the objective lens in the solution and let it approach the slice slowly. Remove all air bubbles trapped on the tip of the objective lens.
Find regions of interest in cleared tissues using epifluorescence. To set image acquisition parameters, first, determine the bit depth for image acquisition as the size of the image increases with the bit. Then set the detection wavelength according to the emission spectrum.
Set the XY resolution, scan speed, and pinhole size. Also adjust the laser power, detector amplifier gain, and offset until a suitable image is obtained. Determine the tiling size based on the size of the ROI, ensuring that the entire length and width of the ROI are captured.
Navigate through the tissue in all planes and set the start and endpoints of the stack. Set the Z step size, according to the desired Z resolution. Collect the images and process them using image analysis software.
Using this protocol, optical clearing of a mouse brain slice of one millimeter thickness was achieved. EGFP expression in the cerebral cortex of PV-FGL mice showed that the fluorescence and structural integrity of the tissues was preserved. Refractive index mismatch induced aberrations caused a noticeable loss of image brightness and resolution of EGFP positive neurons.
These neurons were clearly visualized with the confocal laser scanning microscope when the correction collar was adjusted to match this ScaleS4 solution. 3D reconstruction of EGFP labeled neurons in the mouse primary somatosensory cortex was done in a one millimeter thick brain slice. Individual dendritic arbors decorated with dendritic spines were seen at higher magnification.
Axon terminal arborization and axonal boutons were also observed in the slices. Reflective index matching between imaging fluid and object lens is critical for 3D imaging in cleared tissues. Our technique would facilitate integrating neuron structure from circuit to component scales, providing insights into neuron mechanisms, information of processing in the brain.
