Longitudinal Two-Photon Imaging of Fluorescently Labeled Neurons in a Live Mouse Hippocampus

Take an anesthetized transgenic mouse expressing fluorescent proteins in sparse pyramidal neurons.

The mouse is implanted with an imaging cannula over the dorsal hippocampus.

Position the mouse under the two-photon microscope on a heating pad, securing the cannula’s head plate to the holder to immobilize its head.

Apply ointment to prevent corneal drying.

Clean the cannula with deionized water and inspect it under a low-magnification objective to check for debris, damage, or fluorescence issues.  

Align the imaging cannula with the microscope’s optical axis for precise imaging.

Then, switch to a water immersion high-resolution objective.

Fill the cannula with deionized water, ensuring no air bubbles to avoid image distortion.

Perform two-photon longitudinal imaging using infrared light to selectively excite fluorophores in the focal plane, enabling deep-tissue imaging. 

The implanted cannula facilitates repeated access to the dorsal hippocampus to track dendritic changes over time.

Position the mouse under the microscope over the heating carpet and secure the head plate to the holder. Apply eye ointment to the animal's eyes. Then clean the imaging cannula by using a syringe and a thin needle to drop deionized water into the cannula, followed by removal with a vacuum pump.

Use low-magnification, long-working-distance objectives to visually check the cannula for residual water, dirt, integrity, and the presence of fluorescence. Then align the cannula to the optic axis by adjusting the angles of the head-holder arms.

Switch to a 25x objective with a 1.0 numerical aperture and a 4-millimeter working distance. Then add enough deionized water to the cannula to fill it and maintain excess water on top of the cannula. Finally, use two photon excitation and image the fluorescent signals.