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.

To begin imaging, first set the two-photon laser wavelength. For RFP excitation, use a wavelength of 1,000 nanometers. Wipe the surface of the cover slip with 70% ethanol. Attach the anesthetized pup to the titanium plate on the imaging stage using the titanium bar. Use the goniometer stage to adjust the head such that the cover slip is parallel to the objective lens.

Maintain the body temperature of the pup during imaging, using a heating pad set to 37 degrees Celsius, and reduce the isoflurane concentration to 0.7% to 1%. Place the imaging stage under the 20X objective lens of the two-photon microscope. Apply one drop of water onto the cover slip.

Set the software to acquire z-stack images at 1.4-micron intervals. For layer four neuron imaging, set the z width to between 150 and 300 microns to image the entire dendritic morphology. Use slow scanning and averaging to get clear images showing the neuronal morphology.

It usually takes more than 20 minutes to acquire the entire dendritic morphology.