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.