In Vivo Imaging of Retinal Microglia in a Mouse Model of Glaucoma

Take an anesthetized transgenic mouse with glaucoma, a condition that damages the optic nerve and leads to vision loss.

The microglia, resident immune cells in the eye's retina, express a fluorophore-tagged protein for visualization.

Resting microglia display a ramified morphology with branched projections. Glaucoma-induced nerve damage activates microglia, which adopt an amoeboid shape with fewer projections.

Place the mouse on an ophthalmoscope platform and align the objective lens with one eye.

The eyes are covered with contact lenses to minimize dryness, and the pupils are dilated to expand the imaging field.

Visualize the retina using infrared illumination, which reduces tissue damage.

Identify the optic nerve head, where retinal ganglion cell axons converge to form the nerve.

Utilize retinal blood vessels as landmarks to establish the focal plane.

Switch to fluorescence mode to image the microglia, which appear as brightly fluorescent spots, and evaluate the presence of activated microglia comprising enlarged cell bodies and reduced branching complexity.

Start the imaging session by collecting a fundus view of the inner retina. For this, select the infrared mode, which corresponds to an excitation wavelength of 820 nanometers. Then, adjust the laser power to 100%, and the sensitivity to between 40% and 60%.

Next, working at high speed, locate the eye using the joystick to orient the ophthalmoscope, and prepare to collect a fundus view of the optic disk and retinal vasculature. Inspect the cornea and lens for injury or opacity. Exclude from the study eyes with defects or injuries that may affect image acquisition. Now, by bringing the objective closer to the eye, locate the optic disk area, which is the surface of the optic nerve head, or ONH.

Then, center the image on the ONH. Positioning the ONH correctly is key to obtaining an even focus and emission across the image. The next step is to visualize the inner planes of the retina, as well as the ONH. For a reference focal plane, use major blood vessels located on the vitriol surface of the retina, which corresponds to 59 and 60 diopters, or even deeper at 55 diopters in excavated optic disk areas.

Next, adjust the image saturation, using the knob on the touch panel, until a white halo of illumination spans most of the fundus around the optic disk. Then, select the lower saturation that renders a uniform halo, indicative of an optimal contrast for that eye. It may be necessary to realign the camera if dark areas persist.

Now, collect a high-resolution fundus image of the central retina by averaging 30 frames, taken in real time, to improve the signal-to-noise ratio. This corresponds to 4.7 frames per second normalized. Immediately, and at the same position, prepare to collect a fluorescence image of the GFP positive cells. Switch to fluorescence imaging mode in the touch screen panel, which selects the blue laser with 488-nanometer laser excitation, and a 460 to 490-nanometer barrier filter set. And set the acquisition to 100% laser power, and 100 to 125% sensitivity.

Now, collect a single XY point bidimensional fluorescence image of the ONH, averaging 100x scans. Next, capture a multi-point image of the retina around the ONH. Select composite in the control panel, and then pan with the ophthalmoscope across the nasal temporal axis.

The software automatically averages newly scanned areas and stitches them in real time. If the image quality of areas of the retina is insufficient to allow averaging, the green circle that identifies an area being scanned will become red. The resulting composite image covers an area of up to 1.7/4 millimeters.

Care should be taken during acquisition of the composite image. Images at high resolution can be lost by moving the objective too quickly before completion of scan averaging and image stitching.

When the anesthesia begins to wear off, the imaging session must end because the mouse starts to breathe heavily and imaging becomes impracticable.