This method of in vivo imaging of retinal cells allows for investigations of ophthalmological diseases and understanding normal functions of the retina. This method provides a straightforward and highly reproducible approach to in vivo imaging of the retina by two-photon microscopy. Properly stabilizing the mouse is key to obtaining quality in vivo images.
Practice setting the mouse in the head holder, and become comfortable with the procedure before trying to acquire images. Sedate the animal according to the approved animal use protocol. Apply pupil dilation solution, and leave the mouse in the dark for five minutes to allow the pupils to dilate.
Secure the lower ear canal pin in the inward extended position and the upper ear canal pin in the withdrawn position. Rotate the main arm of the head holder until the earpiece bar is tilted at a 60-degree angle below horizontal. With the mouse facing the bite bar, mount one ear onto the extended lower pin with the pin inserted into the ear canal.
After loosening the screw securing the upper ear canal pin, extend the pin into the other ear canal, and retighten the screw to secure the head. Make sure the mouse is secure in the head holder. Press down on the top of the head.
The mouse should stay securely in the ear bars, and its head should rotate freely around the axis of the ear canals. With the bite bar positioned toward the head of the mouse, gently elevate the head of the mouse to allow the maxillary incisors to be lowered into the bite bar holder, and retract the bite bar with gentle force to secure the head. Use the screw to secure the bite bar into position, and place the mouse and holder onto the microscope stage.
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 eye gel without touching the cornea, and adjust the stage in the X-Y 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 X-Y 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 three. 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 X-Y 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.
When all of the images have been acquired, disable the PMTs and the laser shutter. Switch the excitation light path to epifluorescence illumination and emission light path back to the eyepiece. Then, exit the image acquisition software, turn off the laser in the computer interface, and shut down the hardware in reverse startup order, with the exception of the always-on equipment.
At the end of the analysis, remove the mouse from the head holder, and use a lint-free tissue to gently remove the eye gel. Then, apply lubricant eye ointment to both eyes, and place the mouse onto a 37-degree Celsius warm water circulating heating pad with monitoring until full recumbency, before returning the mouse to its housing. In VGlut-2-Cre mice, retinal ganglion cell somas are clearly discernible and axon fascicles are often apparent.
The trajectory of axons and the negative image of the vasculature facilitates identification of the optic nerve head in VGlut-2-Cre mice, which is useful as a landmark in chronic imaging experiments. In contrast to retinal ganglion cells, amacrine cell neurites are more often observed in the inner plexiform layers. One day after intraocular NMDA injection, retinal microglia demonstrate short processes or amoeboid morphology in accordance with previous reports.
The delivery of Evans blue dye via a single intraperitoneal injection 30 to 60 minutes before imaging induces a strong labeling of the blood vessels emanating from the optic nerve head that persists for at least seven days. After fixation, the true distance between cell pairs within flattened retinal whole mounts can be measured in confocal scans and matched with in vivo pixel distances to determine the average pixel size of in vivo images. Fluorescent microspheres injected into the eyes of mice allows measurement of microsphere diameter in vivo, giving a slightly larger pixel size estimate but with more variance.
In vivo imaging can be paired with histological analyses like immunostaining and in situ hybridization. These methods can identify specific cell types and examine gene expression of imaged cells.
