JoVE Encyclopedia of Experiments
Neuroscience
0 views • 2:42 min • June 17th, 2025
Take an anesthetized transgenic mouse expressing fluorescent proteins in microglia. Secure it on a stereotaxic frame with a motorized stage under a two-photon microscope.
The skull is fitted with a glass-bottom metal tube that gently flattens the alveus above the hippocampal cornu ammonis 1 or CA1 region, ensuring a clear optical interface without
excessive pressure on the underlying tissue.
Fill the metal tube with water to optimize optical transmission.
Activate the pulsed laser at the excitation wavelength.
Using fluorescence from the brain parenchyma and reflected light from the metal tube, adjust the focus on the CA1 region.
Reposition the mouse’s head to align the glass bottom parallel to the imaging plane, ensuring uniform focus across the field of view.
Adjust the objective for optimal resolution and observe in vivo microglial dynamics in the CA1 region.
Next, Image the dentate gyrus, where fluorescent microglia confirm CA1 integrity.
Set the mouse in the head holding device under the objective lens of the two-photon microscope and on the motorized XY scanning stage. Then fill the space between the glass bottom and the objective lens with water without creating air bubbles. Adjust
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