This article describes a method for imaging cleared mouse brains using confocal microscopy. The technique allows for high-resolution visualization of neuronal networks and cellular morphology.
Take a cleared mouse brain with cells expressing fluorescent proteins and lipids removed for transparency for deep-structure imaging with high resolution.
A hydrogel mesh preserves biomolecules and maintains neuronal networks.
Transfer the brain into an imaging chamber containing an agarose ring for support.
Secure the tissue with adhesive and fill the chamber with a refractive index matching solution to minimize light scattering and create a stable imaging environment.
Place the chamber on a confocal microscope stage and gently lower the objective into the solution to establish a continuous column of contact.
Use epifluorescence to locate regions of interest. Then, adjust the laser and imaging parameters to optimize image quality.
Capture a series of z-stacks to create a three-dimensional representation of the tissue.
Use appropriate software to analyze cellular morphology, including dendritic arbors, their spines, and the distribution and density of neurons.
To construct a large tissue imaging chamber for tissues with more than a 5 millimeter thickness, use a 10 centimeter glass dish with a high wall. Place a 50 milliliter conical tube in the center, making sure that the diameter of the tube is large enough to accept the barrel of the objective lens used. Make 3% agarose in water and pour it in the space between the glass dish and the conical tube.
Then allow it to cool for one hour. This will form a ring of solid agarose. Securely adhere the tissue to the bottom of the chamber using superglue, and fill the chamber with refractive index matching solution, which may start to polymerize unless preserved from air and stored at 4 degrees Celsius. Acquire the image using a confocal microscope.
Turn on all the relevant imaging equipment. Place the sample on the stage. Carefully approach the immersion media with the objective and form a continuous column of media. Using epifluorescence, find an appropriate imaging field. Start by setting the resolution and scan speed settings.
If imaging using a confocal microscope, fully close the pinhole to obtain the smallest optical section and thus the best Z resolution. Gradually increase the laser power or sensor gain until a suitable image is obtained with a high signal to noise ratio. If utilizing standard EGFP or tdTomato two color imaging, set the light collection settings.
Set the Z stack parameters based on the observed start and endpoints of the tissue. Set the step size based on the desired Z resolution. Smaller step sizes will yield a greater Z resolution, but may lead to sample bleaching.
When satisfied with the image acquisition settings, acquire the image. Ensure that the image has a high signal to noise ratio, and shows distinct boundaries of structures.
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