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 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.
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.
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.
2. Baseline Imaging of the Ocular Fundus
3. Setting up qAF Imaging
4. Image Acquisition
Note: CRITICAL STEP: The goal in image acquisition should be to obtain 2 high quality 9-frame image stacks per session to control for variability between images within a session. After repositioning the patient and camera, obtain a second session of two images to assess and control for variability. All images will ultimately be calibrated to the internal reference (described below).
5. Image Analysis
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 in well-acquired images. Figure 1B demonstrates a corresponding heat map representation of the qAF levels from Figure 1A. Cooler colors correspond to areas of lower intensity while warmer colors correspond to areas of higher intensity. Maximum intensity is generally seen in the second concentric 8-segment ring (indicated in Figure 1B). This region is also less subject to imaging related variability than regions closer to the image borders, and is outside of the central region where macula pigment has a large impact on qAF levels. Thus, the mean intensities of this ring are used for most data analyses 13. Figure 2 presents a representative analysis in an eye with AMD demonstrating geographic atrophy (GA), 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.
Figure 1. Autofluorescence in healthy eye. (A) Autofluorescence (AF) image of the right eye of a normal patient. OD: optic disk, Fo: fovea, Ma: macula, Ref: internal reference. (B) Post-processing qAF map of AF image from (A). Warmer colors correlate to higher AF intensity. Fixed regions are shown, and qAF values of each region are indicated. 8-segment perifoveal ring used in data analysis is indicated by the dashed line. Please click here to view a larger version of this figure.
Figure 2. Autofluorescence in eye displaying geographic atrophy due to AMD. (A) Autofluorescence (AF) image of the left eye of a patient with advanced AMD showing geographic atrophy (GA) of the RPE (representative region circumscribed by dashed line). OD: optic disk, Fo: fovea, Ma: macula, Ref: internal reference. Note markedly reduced and absent AF in regions corresponding to GA in the macula. (B) Post-processing qAF map of AF image from (A). Fixed regions are shown, and qAF values of each region are indicated. 8-segment perifoveal ring used in data analysis is indicated by the dashed line. Please click here to view a larger version of this figure.
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 and monitoring of retinal disease, and eventually in assessing the efficacy of therapeutic interventions, such as drug or gene therapy. qAF may also assist in stratification of individuals at-risk for conditions such as AMD.
We have generated a large normative database of qAF data to be used as a reference tool for the interpretation of retinal pathology 13, and have also described qAF in several disease states, including Stargardt disease 10, bull's eye maculopathy 12 and Best disease 11. In healthy retina, there are different qAF levels between ethnic groups, with significantly higher qAF in whites than blacks and Asians, and demonstrated that women have higher qAF levels than men. Perhaps most strikingly, qAF increases as patients age, which corresponds with RPE lipofuscin levels, as previously measured by spectrofluorometry 14 . Although current normative data only extend up to age 60, it appears that patients have a measured decrease in RPE lipofuscin after age 70 14. Interestingly, it does not appear that changes in RPE cell number occur as patients age 2and so the observed decreases in AF may be due to a redistribution or reduction in RPE lipofuscin 3. It will be interesting to determine whether these decreases in AF in old age correlate with impaired RPE function and increased risk of AMD.
This protocol allows for reliable quantitative results that can be compared longitudinally, between patients, and between cSLO machines. One limitation is that qAF cannot accurately measure AF in older patients with lens opacities, which limits the technique in a subset of this high-risk population. One possible solution is to use a reflectometry method to individually estimate light losses in the media. Fortunately, older patients who are pseudophakic with clear posterior capsules and known intraocular lens transmission characteristics are good candidates for qAF. However, the pupils of elderly patients and those with prior cataract surgery often do not dilate to more than 6mm, which limits light passage and prevents accurate data collection. In order to image such patients, an aperture may be inserted into the cSLO by the manufacturer in order to decrease the diameter of the laser. This modification has been successfully utilized to perform qAF imaging in mice 15. Another limitation is that the technique is challenging to perform and is highly operator dependent. It is essential to consistently obtain high quality images to ensure accurate measurements of AF levels. In order to achieve this, the authors recommend strict adherence to the imaging protocol as well as adequate practice. Although some patients poorly tolerate the bright flashes of light required for AF imaging, assuring patients that exposure levels are well within the safe limits is helpful 1. To allow for optimal imaging, it is critical to dilate pupils to at least 6 mm and to obtain multiple images with unobstructed light transmission (see above). Achieving optimal focus and avoiding oversaturation of pixels are also essential. Helpful practices include communication with the patient while imaging, use of an assistant if the eyelid needs to be lifted and use the foot pedal for triggering the camera, as described above.
In summary, understanding the pathophysiology of the RPE in retinal degenerations remains an area of active research, and of potentially important therapeutic impact. As qAF allows for direct comparison of AF levels in images obtained longitudinally, between patients and between centers, it is a valuable tool that can contribute to this understanding as well as provide useful clinical information. The detailed protocol outlined here will assist others in the acquisition of reliable qAF data, and that the important clinical applications of qAF will encourage research centers and clinical retina specialists to make use of the qAF technique.
The authors have nothing to disclose.
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).
Spectralis HRA + OCT | Heidelberg Engineering | n/a | |
0.5% tropicamide ophthalmic solution | n/a | n/a | Any brand can be used |
2.5% phenylephrine ophthalmic solution | n/a | n/a | Any brand can be used |
Internal fluorescent reference | Heidelberg Engineering | n/a | |
IGOR Pro software | WaveMetrics | n/a |