Two-Photon Microscopy of Astrocytes and Excitatory Neurons in a Cleared Mouse Hippocampal Tissue

Begin with a cleared mouse hippocampal tissue rendered optically transparent by removing lipids. The tissue is embedded in a hydrogel mesh to stabilize biomolecules and preserve cellular structures.

The tissue contains astrocytes expressing red fluorescent protein and excitatory neurons expressing green nuclear protein, enabling distinct visualization. 

Place the tissue on a slide and create a chamber using hot glue, leaving a small gap. 

Apply a refractive index matching solution to hydrate the tissue and prevent bubbles, then seal with a coverslip. 

Fill the chamber with a refractive index matching solution to reduce light scattering and close the gap.

Visualize the tissue using a two-photon microscope. 

Near-infrared light excites the red and green fluorescent proteins at the focal plane, enabling high-resolution imaging with minimal photodamage. Fluorescence emissions are detected separately, clearly revealing the spatial proximity between astrocytes and the excitatory neurons’ nuclei.

First, place the sample in the middle of the slide. Using hot glue, create walls on the edges of the slide, almost as high as the tissue. Make sure to leave a small gap at one of the corners. As the hot glue layer approaches the height of the brain slice, apply one to two drops of the refractive index matching solution on the sample to moisten the upper surface and prevent bubble formation.

While the hot glue is still liquid, seal the top with a coverslip, placing it as evenly as possible. Then, fill the chamber with the refractive index matching solution. Close the gap with hot glue. If the hot glue walls extend beyond the borders of the slide, cut the extending edges. If an oil immersion objective is used for imaging, add another two to three millimeters of glue to the walls above the coverslip to retain the immersion solution. After this protocol, the brain tissue will be cleared.