We would like to thank our collaborators, Francois Delori, Tomas Burke, and Tobias Duncker.
Research Support: NIH/NEI R01 EY015520 (RTS, JPG), and unrestricted funds from Research to Prevent Blindness (RTB).

Ethics Statement: All patients enrolled in these studies were done so in accordance with approved institutional review board oversight at New York University School of Medicine.
1. Patient Selection and Initial Preparation for Imaging
Note: The following materials are required: 0.5% tropicamide ophthalmic solution, 2.5% phenylephrine ophthalmic solution, cSLO equipped with spectral domain optical coherence tomography (SD-OCT), and internal fluorescence reference.
...

<ol>
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STA: no disclosures, JPG: no disclosures, RTS: consultant, Ocata Therapeutics.

Abnormal RPE lipofuscin distribution, whether increased or decreased, is a sensitive marker of retinal disease and is generally associated with loss of sensory retina function. Here, we describe application of qAF for the evaluation of RPE lipofuscin. Incorporation of an internal fluorescent reference to correct for variable laser power and detector sensitivity9 alongside our standardized imaging technique allows for reliable quantitation of AF levels. It is our goal that this method will aid in the diagnosis ...

The retinal pigment epithelium (RPE) supports the function of the sensory retina through numerous processes 1. Age-related macular degeneration (AMD) is the most important cause of untreatable blindness in industrialized countries and is characterized by changes in the RPE, including loss of pigment, loss of function and atrophy. In AMD and in normal aging, the RPE accumulates fluorescent, lysosome-derived organelles containing phagocytosed photoreceptor fragments, referred to as lipofuscin granules. The accumulation of RPE lipofuscin has been thought to indicate oxidative dysfunction 1, but recent studies have shown that the RPE morphology remains normal in aged eyes with high lipofuscin levels 2. However, abnormal patterns of lipofuscin distribution, in particular loss of lipofuscin, are documented markers for AMD and AMD progression, both histologically and clinically 3,4
Defective processing of RPE lipofuscin has also been shown to occur in certain inherited retinal degenerations. Patients suffering from Stargardt disease (STGD) accumulate lipofuscin in the RPE at a young age, eventually developing vision loss similar to that seen in AMD 5. These findings suggested that lipofuscin accumulation may itself be toxic and drive RPE dysfunction 6,7. However, a detailed imaging study of subjects with STGD over time did not confirm that focal lipofuscin accumulation led to subsequent RPE loss 8. Hence, although lipofuscin abnormalities are markers for retinal degenerations, a role for direct toxicity of lipofuscin remains unproven.
The RPE is the most posterior cell layer of the retina, but generates the majority of fluorescent signal from the ocular fundus. Generation and detection of autofluorescence (AF) derived from the RPE can be performed using confocal scanning laser ophthalmoscopy (cSLO), which allows for visualization of the spatial distribution of fundus AF. Certain retinal degenerations demonstrate distinctive patterns of fundus AF, and AF imaging aids in the diagnosis and monitoring of these conditions. Although standard AF imaging is clinically important, quantitative AF (qAF) has become an important means of assessing RPE health. We and others have developed a standardized approach that can reliably determine qAF levels at specific retinal locations 9. qAF has potential applications in the diagnosis and monitoring of retinal conditions, and may also have utility in prognosis and risk stratification. In addition, the diagnostic capabilities of qAF have also been described for certain retinal disorders 10-12. Here, we provide step-wise details for performing our technique accompanied by a visual demonstration of its application in the evaluation of healthy and diseased eyes.

<table border="0" cellpadding="0" cellspacing="0" width="671">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:243px;">Spectralis HRA + OCT</td>
			<td style="width:143px;">Heidelberg Engineering</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">0.5% tropicamide ophthalmic solution</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2.5% phenylephrine ophthalmic solution</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:243px;">Internal fluorescent reference</td>
			<td style="width:143px;">Heidelberg Engineering</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IGOR Pro software</td>
			<td style="width:143px;">WaveMetrics</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative fundus autofluorescence for the evaluation of retinal diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

This technique was used to study qAF in both healthy 13 and disease states 10-12. In healthy eyes (Figure 1), AF emitted from the RPE is distributed relatively uniformly throughout the fundus (Figure 1A). Reduced intensity is seen in the central macular region due to blocking of light by macular pigment, and at the sides and corners of the image due to the optics of the eye and camera. Vessels appear dark and should be in clear focus...

Watch this Scientific Journal Video about Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases at JoVE.com

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

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 retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

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.

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11.3K Views.  NYU School of Medicine. 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 ...