Two-Photon In Vivo Imaging of the Mouse Retina

Take an anesthetized transgenic mouse with its head secured at an angle on a holder for in vivo imaging of the mouse retina.          

The retina contains retinal ganglion cells, or RGCs, expressing a fluorophore-tagged protein.

Apply eye lubricant and mount a coverslip over the eye.

Under a microscope, uniformly illuminate the retina to excite the fluorescent proteins in the RGCs.

Using the emitted fluorescence, obtain a widefield view of the focal plane RGCs and out-of-focus RGCs.

Switch to two-photon imaging mode, focusing low-energy near-infrared light into the RGC layer.

At the focal point, the combined energy from two photons excites the fluorophore, which then releases energy as fluorescence. 

As the excitation is confined to a single point, fluorescence from other focal planes is minimized.

Capture images at multiple focal planes along the z-axis to image the entire RGC layer.