Two-Photon Microscopy for Imaging Neuronal Morphology in a Mouse Pup

Begin by setting up the excitation light for the two-photon microscope.

Take an anesthetized transgenic mouse pup with a circular glass imaging window and a head plate implanted in its skull.

The neurons express red fluorescent proteins to facilitate imaging.

Clean the glass imaging window and secure the pup onto the two-photon imaging stage.

Align the imaging window with the microscope objective by adjusting the pup’s head.

Use a heating pad to maintain the pup's body temperature.

Adjust the anesthetic flow rate.

Apply a drop of water onto the coverslip, then lower the microscope's objective lens.

Two-photon microscopy uses two photons of light to activate fluorescent proteins, allowing detailed neuronal imaging with minimal background noise and photobleaching.

Capture images of the neurons at various depths to visualize their structure

Finally, stack the images to generate a three-dimensional representation of the neuron, including their neuronal projections, to study growth-related morphological changes.