The overall goal of the following experiment is to image fluorescently labeled intact biological samples without histological sectioning. This is achieved by sequential incubation of fixed tissues in organic solvents until a full transparency is achieved. As a second step.
The transparent tissues are imaged in 3D using a laser scanning microscope. Next, the imaging results are analyzed and the neuronal structures in the un sectioned spinal cord and brain can be reconstructed in 3D from the collected three disco images. This method can help answer key questions in the neuroscience field regarding how to map neuronal and GAL connections in the entire brain and spinal cord.
The implications of this technique extend towards the therapy of neurodegeneration as we can now visualize neural integrity throughout the entire nervous system. Though this method can provide insight into the organization of the nervous system, it can also be applied to other organs such as the lung or tumor tissue. Once mastered, this technique takes only a few hours for small organs such as the spinal cord or the lung, and about one or two days for the brain Begin the procedure by adding two to three milliliters of freshly prepared 50%THF to one glass vial per harvested tissue.
Then transfer the fixed organs into the clearing solution and securely close the vials. Place the vials on a rotator and cover them in aluminum foil, stirring the samples for the appropriate time at a constant speed of 30 RPM. Then remove the clearing solution and use a new PE or pipette to suspend the tissue in the next solution.
As indicated in the table during the final clearing step, keep the samples in solution until they become completely transparent for multi photon or confocal microscopy. As soon as a complete clearing is achieved, mount the sample on an imaging slide to this end. Use dental cement to make a border around the tissue.
Fill the pool with the final clearing solution just before the dental cement becomes fully rigid, and then immediately place the cleared sample in the middle of the pool and cover it with a cover glass. Press the cover glass until it is completely sealed by the dental cement and touches at the surface of the cleared organ for light sheet microscopy as soon as a complete clearing is achieved mount, and then manually turn the screw of the sample holder to fix the sample. Next, dip the sample into the imaging chamber of the light sheet microscope filled with the final clearing solution appropriate for the imaged tissue.
Then collect Z scans covering the entire cleared tissues at the best resolution that the microscope can deliver, zooming in on the regions of interest to collect higher resolution images. After collecting the images, load the image series in the Amira software with the resolve RT module. When the series has been loaded, enter the correct voxel sizes in the image read parameter window and click okay.
Next, select the multiplanar view sub application. Then use primary and thickness sliders to choose the optimum projection thickness and threshold for the gray values. Finally, use the crop corner module to browse the slices in different 3D orientations.
To visualize the samples, Mapping the structural organization of neurons is extremely important for understanding how they function in health and disease. While three disco can be employed on various organs, it is then particularly useful for tracing long neuronal connections in the spinal cord and brain. For example, using ultra microscopy scans of large cleared spinal cord segments, the axonal connections can be followed over centimeters in the rodent spinal cord as illustrated in these images.
In a similar way, the entire cleared mouse brain and hippocampus can be imaged to follow neuronal connections in the brain. When confocal or multi photon microscopy is used, the imaging resolution can be significantly improved, especially in the Z dimension. For example, multi photon imaging of cleared spinal cords from GFPM line mice achieves a seamless image throughout the entire 1.5 to two millimeter depth of the spinal cord.
Confocal microscopy on the cleared spinal cord delivers improved resolution in a similar way. Multi photon microscopy imaging of cleared brains delivers very high resolution images to visualize fine details of neuronal structures, including dendritic spines. The samples for three disco can be labeled in various ways.
For example, it is possible to label the entire vasculature of the brain and spinal cord using lectin conjugated fluorescent tracers, which can be used to study the blood brain barrier in health and disease. Using three disco smaller cells in large tissues can also be imaged. For example, both microglia and astrocytes are highly implicated in the pathology of neurodegeneration, including Alzheimer's disease and traumatic injuries.
Using three disco, their density and distribution in the spinal cord can be studied. Non neuronal tissues can also be imaged. For example, Clara cells in the entire rodent lungs can be immuno labeled with antibodies and imaged without sectioning at single cell level.
Similarly, it is possible to clear and image cells in the unsanctioned pancreas tissue. For example, the fluorescently labeled alpha cells are imaged after clearing in these pancreas images. Visual demonstration of this method is important because while the clearing and imaging steps are straightforward, the data analysis can be complicated.
While applying this procedure, it's important to image clear tissues as soon as the complete transparency is achieved because the fluorescent signal will degrade over time in the clearing solution. The development of this technique paved the way for researchers to explore details and intact organs, such as mapping neuronal networks in the brain. After watching this video, you should have a good understanding of how to clear and image cellular and subcellular structures in the entire organs, and subsequently visualize and analyze them.
