In Vivo Two-Photon Imaging of Microglial Dynamics in the Mouse Hippocampus

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 the focus, to CA1 with the guidance of fluorescence emitted from the brain parenchyma and the reflected light from the edge of the metal tube, under continuous illumination by the pulsed laser.

Adjust the angle of the mouse head by tilting the head holding device so that the glass bottom is parallel to the imaging plane. Then, adjust the correction collar of the objective to achieve the highest resolution at a depth of the target structure in the CA1. Next, confirm that the fluorescent cells in the molecular layer of the dentate gyrus, or DG, at a depth of 500 micrometers from the glass bottom, are imaged in the entire field of view. Only in case of a successful surgery, the DG signals can be detected immediately.

Ensure that microglia have recovered their ramified morphology. Then perform in vivo imaging on the layer of interest in the CA1.