The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.
The overall goal of quantitative autofluorescence, or qAF, is to evaluate the overall level of lipofuscin in the support cells of the retina, the retinal pigment epithelium, or RPE, in both healthy and diseased eyes. This technique can help answer key questions in retinal cell biology, such as how the health of the retinal pigment epithelium interacts with the development of age related macular degeneration, or AMD. The main advantage of this technique is that it allows for the determination of absolute levels of lipofuscin autofluorescence in the fundus, and therefore quantitative and reproducive follow up between patients, between patient imaging sessions, and in large study populations.
Prior to imaging, appropriately set up confocal scanning laser opthamoscopy, or CSLO, for data acquisition according to the manufacturer's instructions. To mount an internal fluorescent reference, which is purchased from the manufacturer, twist the lens to remove it, unscrew the machine's metal ring, and replace it with the new metal ring containing the reference. It is essential for pupils to be dilated to at least 6mm for uninterrupted passage of light.
With 0.5%tropicamide, and 2.5%phenylephrine, dilate the patient's pupils at least 6mm, which is essential for uninterrupted passage of light to see and to measure the fundus. Properly position the patient on the CSLO with chin resting on the chinrest, forehead placed against the forehead rest, and lateral canthi properly aligned with the indicators. To acquire baseline images, first image the fundus with near-infrared reflectance, or IR light, in order to centralize the camera over the macula and obtain rough focus.
With the patient properly positioned, switch the hardware setting on the control panel to IR imaging mode, manually position the camera until the fundus is in full focus, and take an image. Adjust the setting on the control panel to IROCT, which uses spectral domain optical coherence tomography, or SDOCT, in conjunction with IR imaging, to evaluate the macula for underlying disease. Use the guides present in the imaging window to correctly orient the OCT to the IR image of the fundus.
To achieve optimal SDOCT quality, position the camera such that the OCT image is in the top third of its imaging window. Acquire at least one horizontal line scan through the fovea, and spanning the entire imaging field. To set up qAF imaging, use high speed image acquisition, which decreases risk of signal loss due to patient movement, and blockage of light by iris or eyelids.
Use Mean of 9 frames, which rapidly and sequentially captures nine image frames that are then averaged to reduce noise and artifact. Choose a 30 x 30 degree field, which refers to the degrees of retinal area captured during image acquisition. After warning the patient about the blue light, turn on the AF mode and align the camera axis so that the screen is maximally filled with fundus AF, with minimal darkening of sides and corners of image.
If patients have difficulty tolerating the bright blue light, move the camera further away from the eye to start imaging, and slowly bring the camera toward the patient until the fundus is in full view. Either manually or with the opthamoscope joystick, adjust the camera alignment by moving the CSLO to reposition the camera such that the AF signal is at its highest level throughout the field, rather than achieving the sharpest image. During image acquisition, colored pixels visible in either the internal reference or the fundus indicate over saturation, and thus loss of signal.
Fully expose the retina to AF light for at least 20 seconds to minimize the absorption of light by rhodopsin in the sensory retina during imaging. Use this period to optimize camera alignment, focus, and sensitivity. Have the subjects blink before each image acquistion, as a fresh tear film improves signal quality.
Avoid eyelids in the plane of acquistion and take the image. It's important to generate at least two high quality images per imaging session to have for data analysis. Perform post-image processing by using the CSLO software to compute the mean of the nine frame stack, to increase the signal to noise ratio.
Carry out image analysis according to the text protocol. As shown here, in healthy eyes, AF emitted from the RPE is distributed relatively uniformly throughout the fundus. Reduced intensity is seen in the central macular region due to the blocking of light by macular pigment, and at the sides and corners due to the optics of the eye and camera.
Vessels should be dark and in clear focus. This figure represents a corresponding heat map presentation of the qAF levels from this figure. Cooler colors correspond to areas of lower AF intensity, while warmer colors correspond to areas of higher AF intensity.
Maximum intensity is generally seen in the second concentric eight segment ring, and the mean intensities in this area are used for most data analysis. Shown here is a representative analysis of the eye with AMD, demonstrating geopgraphic atrophy, an advanced form of AMD. This form of AMD results in localized areas of RPE loss, evidenced by markedly reduced or absent AF, and causes progressive central vision loss.
While attempting this procedure it's important to remember to select candidate patients carefully and obtain high quality images reproduceably to maximize data quality. Use of this procedure can allow for the longitudinal follow up of retinal health to determine the progress of retinal degenerations. After its development, this technique paved the way for researchers in the field of opthamology to follow and analyze retinal degenerations.
After watching this video, you should have a good understanding of how to use quantitative autofluorescence to evaluate your patients with retinal degenerations. Thanks for watching, and good luck with your experiments.
