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.

Apply lubricant eyedrops to both eyes of the mouse. Rotate the main arm of the head holder until the pupil of one eye is oriented in line with the light path, and place a number 1.5 coverslip into the compact filter holder. After securing the holder to the microscope stage, lower the coverslip until it contacts the lubricant i-gel without touching the cornea. And adjust the stage in the xy dimension and the objective z position until the widefield excitation light fully covers the cornea.

Use the eyepiece to continue to adjust the z position, until the fluorescent cells or structures in the retina come into focus, increasing the epifluorescence illuminator as necessary to resolve individual cells or structures of interest. Fine-tune alignment of the retina with the imaging light path. Adjust the head angle until only expansion or contraction of the out-of-focus light occurs when changing the focal plane, minimizing xy distortions.

Then turn off the epifluorescence illuminator and close the illuminator shutter. For two-photon imaging, follow all institutional laser safety protocols. Turn off and cover all ambient light and switch the excitation light path to the laser and the emission light path to the PMTs.

In the image acquisition software, set the frame size to 512 by 512 and the frame average to 3. Set the z-stepping to start at the top of the stack and progress downward, minimizing the two-photon laser activation of the photoreceptors. Turn on and enable the PMTs, and adjust the voltage to 680 volts.

Enable the imaging and emission shutters. Begin a live image preview of the target tissue starting at a 1% laser power, and auto-adjust the display brightness to visualize the cells or structures of interest. If the target tissue is dim or unclear, increase the laser power percentage until structures become visible without surpassing 45 milliwatts.

Maneuver the microscope stage in the xy direction to center on a desired imaging area, and navigate to the z plane, with the structures of interest in focus. For a chronic time-lapse experiment, open a previously obtained image to use as a reference for the cells of interest, taking care that the angle of imaging in the current image is similar to that of the previous images. Then navigate to the upper and lowermost z planes of interest to set the z limits of the imaging stack and acquire the image.