Fundus Autofluorescence Imaging: Do You Need It and When?

STEFFEN SCHMITZ-VALCKENBERG, MD · FRANK HOLZ, MD

Fundus autofluorescence (FAF) imaging using a confocal scanning laser ophthalmoscope (cSLO) was initially described by von Rückmann et al. in 1995.¹ Pioneering work on the spectral analysis of the origin of the autofluorescence signal was performed by Delori and coworkers in parallel.² In 2003, Spaide introduced a modified fundus camera (using longer wavelengths than originally applied) for FAF imaging and, recently, near-infrared autofluorescence imaging was described.^3-5 Various studies on FAF imaging have been performed in the meantime and, subsequently, this imaging technique is being increasingly used for research purposes. Today, FAF imaging is at the doorstep to a broader application as a noninvasive clinical imaging tool.

In our overview on FAF imaging we address pertinent issues including refined phenotyping of retinal and macular disease and monitoring of disease progression and therapeutic responses, as well functional correlations of FAF findings.

WHAT IS FUNDUS AUTOFLUORESCENCE?

Fundus autofluorescene imaging is an in-vivo imaging method for metabolic mapping of naturally occurring fluorophores of the ocular fundus. The dominant source are fluorophores, such as A2-E (N-retinylidene-N-retinylethanolamine) in lipofuscin, that accumulate in the retinal pigment epithelium (RPE) as a byproduct from the incomplete degradation of photoreceptor outer segments.^2,6,7 The topographic distribution of FAF intensities is altered in the presence of excessive accumulation or loss of lipofuscin/RPE cells. Additional intrinsic fluorophores may occur with disease in the outer retina and the subneurosensory space. Minor fluorophores, such as collagen and elastin in choroidal blood vessel walls, may become visible secondary to RPE atrophy. Finally, pathological alterations in the inner retina at the central macula, where the FAF signal is usually partially masked by luteal pigment (lutein and zeaxanthin), may result in manifest variations in FAF intensities.

Figure 1. Idiopathic juxtafoveal telangiectasia Type 2a shown by fundus photography (A), fundus autofluorescence (B) and early- (C) and late-phase (D) fluorescein angiography. No obvious alterations are visible by fundus photography. Fluorescein angiography shows telangiectatic vessels in the parafovea and late-phase hyperfluorescence. Fundus autofluorescence imaging reveals markedly increased levels of the central fovea as opposed to the typical decreased signal in normal subjects.

Recording of FAF is easy, requires little time, and is noninvasive. FAF signals are emitted across a broad band from 500 to 800 nm². Excitation is usually induced in the blue range (λ = 488 nm), and a barrier filter above 500 nm is used to detect emission of the autofluorescence signal.⁸ The intensity of the signal is about 2 orders of magnitude lower than the background of a fluorescein angiogram. In the presence of nuclear lens opacities, the yellowing of the lens may lead to relevant absorption of the excitation light. The confocal optics, together with the enhanced sensitivity of the cSLO and averaging of several individual scans following automated alignment, ensure the acquisition of meaningful images in most patients.

IDENTIFICATION OF EARLY DISEASE STAGES

Fundus autofluorescence imaging may allow for identification of retinal diseases when they are not otherwise evident. Metabolic changes at the level of the photoreceptor/RPE complex may not be visualized by funduscopy or other routine imaging techniques such as fluorescein angiography in early manifestations of macular and retinal dystrophies. This is particular helpful when investigating patients with unknown visual loss or a positive familiar history of hereditary retinal diseases.^9,10 Recently, we have shown that FAF imaging reveals distinct abnormalities in idiopathic juxtafoveolar retinal telangiectasia group 2a (Figure 1).¹¹ Already, in the early disease stage, where only subtle dilation of parafoveal retinal blood vessels can be detected by fluorescein angiography, the FAF signal in the central macular is typically increased, which is thought to result from a depletion of macular pigment in layers anterior to the RPE. In early dry age-related macular degeneration (AMD), alterations at the level of the RPE may become visible in areas that appear normal on funduscopy. Reticular drusen as a particular drusen phenotype are readily visible on FAF images.

PHENOTYPING

Alterations in FAF intensities at the outer retina in various retinal diseases are typically much more pronounced with structural or optical alterations seen with other imaging techniques. This helps in diagnosing hereditary retinal disorders, such as Stargardt disease and vitelliform macular and pattern dystrophies, and it may be used for correlation with specific genetic defects.¹⁰ For example, focal flecks in Stargardt disease associated with a markedly increased FAF signal are more readily delineated on FAF images as compared to fundus photographs (Figure 2).¹² Furthermore, it has been demonstrated that distinct phenotypic alterations can be detected in patients that had been diagnosed with AMD. Classification systems for FAF patterns both in early AMD and advanced atrophic AMD have been introduced.^13,14 FAF findings in this context also help to distinguish AMD from late onset of macular dystrophies mimicking age-related changes.

DISEASE MARKERS

Several lines of evidence suggest that excessive lipofuscin accumulation represents a common downstream pathogenetic pathway in various hereditary and complex retinal diseases.^6,15 Clinical observations indicate different degrees and extension of levels of increased FAF that do not or only poorly correlate with findings obtained by other imaging techniques (Figure 3). These FAF changes, remote from visible alterations, may suggest more widespread abnormalities and diseased retinal areas.¹⁶ Focally increased FAF and, therefore, excessive RPE lipofuscin load may indicate dysfunctioning RPE cells. Indeed, the pathophysiological role of abnormal FAF is underscored by recent longitudinal studies. For example, it has been demonstrated that areas with increased FAF signal and, thus, excessive RPE lipofuscin build-up precede the development of new areas or the enlargement of preexisting atrophic patches in geographic atrophy secondary to AMD.¹⁷ Furthermore, the extension of abnormal FAF, as well as the previously introduced FAF pattern classification, has an impact on atrophy enlargement rates over time and may therefore serve as predictive determinants.^18,19 The identification of high-risk characteristics, ie, clinical biomarkers, for disease progression may be helpful not only for monitoring patients with advanced atrophic AMD, but also for performing clinical interventional trials with "fast progressers." In patients with choroidal neovascularization (CNV) or in inflammatory diseases, FAF abnormalities typically extend beyond the angiographically visible alterations, also indicating a more widespread disease.^16,20 Changes in the FAF signal may permit physicians to estimate the extent of damage, diagnose sequella such as secondary CNV, learn more about the inflammatory process, and possibly anticipate future problems caused by disease.

Figure 2. In Stargardt macular dystrophy, fundus pathology is better detected with fundus autofluorescence imaging than fundus photography. Typically, autofluorescence imaging shows a central oval area of reduced signal surrounded by small disseminated spots of reduced and increased intensity. There is also central multifocal RPE atrophy present showing very decreased FAF intensity. Yellowish-appearing flecks on fundus photography correspond to punctate spots with bright autofluorescence signal.

Figure 3. In atrophic AMD, atrophy areas appear as sharply demarcated areas with depigmentation and enhanced visualization of deep choroidal vessels on fundus photograph (left). At the corresponding fundus autofluorescence image (right), atrophic patches are clearly delineated by decreased intensity and high-contrast to non-atrophic retina. Surrounding atrophy, in the junctional zone of atrophy, levels of marked FAF intensity are observed which are invisible on fundus photography. These abnormalities tend to precede atrophy over time and may serve as disease markers.

MONITORING OF DISEASE PROGRESSION

Due to the absence of RPE lipofuscin, atrophic areas such as in atrophic AMD or Stargardt disease, exhibit a markedly reduced signal. Such diseased areas and atrophic patches can be easily identified and, moreover, precisely quantified by customized image-analysis software.^21,22 This allows for noninvasive monitoring of atrophy progression over time (Figure 4).

Figure 4. Monitoring of atrophic progression over time with fundus autofluorescence imaging, showing the natural course of the disease over 5 years. Note, the preserved foveal island ("foveal sparing") in the center of the central atrophic patch, which becomes smaller during the review period.

FUNCTIONAL CORRELATION

The relevance of alterations in FAF images can further be addressed by assessing corresponding retinal sensitivity. Severe damage to the RPE, such as atrophy, melanin pigment migration, or fibrosis leading to compromised photoreceptor function as confirmed by microperimetry, is topographically confined to decreased autofluorescence.^23,24 In patients with geographic atrophy secondary to AMD, it has been shown that — in addition to absence of retinal sensitivity over atrophic areas — retinal function is relatively and significantly reduced over areas with increased FAF intensities compared to areas with normal background signal.²³ Localized functional impairment over areas with increased FAF has also recently been confirmed in patients with early AMD. Using fine matrix mapping, it has been demonstrated that, interestingly, rod function is more severely affected than cone function over areas with increased FAF in patients with AMD.²⁴ These studies are in accordance with the observation of increased accumulation of autofluorescent material at the level of the RPE prior to the occurrence of cell death. As normal photoreceptor function is dependent on normal RPE function, in particular with regard to the constant phagocytosis of shed distal outer segment stacks for photoreceptor cell renewal, a negative feedback mechanism has been proposed: Cells with lipofuscin-loaded secondary lysosomes would phagocytose less shed photoreceptor outer segments, subsequently leading to impaired retinal sensitivity. This would also be in line with experimental data showing that compounds of lipofuscin such as A2-E possess toxic properties and may interfere with normal RPE cell function.¹⁵

In patients with retinitis pigmentosa and cone dystrophies, parafoveal rings of increased FAF have been noted that tend to shrink or enlarge with disease progression, respectively (Figure 5).^25,26 Interestingly, functional testing by microperimetry and electrophysiology indicate that these rings demarcate areas of preserved photoreceptor function. In retinitis pigmentosa, a gradient loss of sensitivity is present outside the arc of the ring with increasing eccentricity.

FUTURE APPLICATIONS FOR THERAPEUTIC INTERVENTIONS

The integrity of the RPE may play an important role in the development and evolution of CNV, photoreceptor viability, and, subsequently, response to therapeutic intervention. One recent study indicates that FAF changes better correlated with visual acuity than symptoms length and lesion size.²⁰ While early CNV typically manifested with patches of "continuous" or "normal" autofluorescence (corresponding with areas of hyperfluorescence on the comparative fluorescein angiograms), longstanding CNV exhibited more areas with decreased intensity. The latter observation was explained by photoreceptor loss and scar formation with increased melanin deposition. Of note, a decreased FAF signal does not necessarily mean irreversible retinal damage, as it might also be caused by subretinal fluid or hemorrhages. The theoretical concept of assessing RPE integrity and viability to determine select patients who will actually benefit from therapeutic interventions such as intravitreal anti-vascular endothelial growth factor treatment is attractive and warrants further investigation.

In patients with advanced atrophic AMD, FAF imaging may also helpful to develop and assess new emerging therapeutical strategies. Visual cycle modulators, which aim to target the detrimental accumulation of toxic lipofuscin in the RPE, appear as appealing pharmaceutical agents to slow down the progression of atrophy. One prominent agent is fenretinide (N-[4-hydroxyphenyl]retinamide), an oral compound that has been shown to lower the production of toxic fluorophores in the RPE in a dose-dependent manner in the albino ABCA4 -/- mice.²⁷ This vitamin A derivate acts by competing with serum retinol for the binding sites of retinal-binding protein and promotes renal clearance of retinol. The bioavailability of retinol for the RPE and photoreceptors is consequently reduced and less toxic retinoid byproducts such as A2-E may be generated. A phase 2, randomized, double-masked, placebo-controlled multicenter study that aims to include over 200 GA patients, was initiated in the United States in 2006 by Sirion Therapeutics, Inc. (Tampa, FL). The therapeutic concept of fenretinide is underscored by previous FAF findings. To reduce the observational period in a slowly progressive disease, to minimize the sample size, and to better demonstrate possible treatment effects, the patient recruitment in this study involves the identification of high-risk features in this first large interventional trial in patients with geographic atrophy secondary to AMD.

Figure 5. This patient with retinitis pigmentosa shows the typical ring of increased autofluorescence in the parafovea, which is not visible on fundus photography. Functional testing reveals that this ring correlates with functional abnormalities and represent a demarcation line between normal and abnormal functional retina.

In retinal dystrophy, FAF imaging may be used to assess the functional preservation of the outer retina and would therefore have implications for future treatment. In patients with Leber congenital amaurosis having vision reduced to light perception and undetectable electroretinograms, normal or minimally decreased FAF intensities have been reported.²⁸ This suggests that the RPE/photoreceptor complex is, at least in part, functionally and anatomically intact and would indicate that photoreceptor function may still be rescuable.

CONCLUSIONS

FAF imaging gives information over and above conventional imaging techniques in various retinal disorders and, thus, adds to our armamentarium to diagnose and manage patients. It is not time-consuming, easy to perform, and a noninvasive imaging method. Particularly in disorders of the outer retina, the application offers the opportunity to identify disease-related abnormalities and to determine the integrity of the RPE. Notably, it represents not just a technique to visualize structural changes, but actually allows for metabolic mapping and correlation with retinal function. Several studies have demonstrated its clinical relevance, while there are numerous promising future applications. In combination with other emerging technologies such as spectral-domain optical coherence tomography, FAF imaging may considerably add to our understanding of retinal disease. RP

REFERENCES:

1. von Ruckmann A, Fitzke FW, Bird AC. Distribution of fundus autofluorescence with a scanning laser ophthalmoscope. Br J Ophthalmol. 1995;79:407-412.
2. Delori FC, Dorey CK, Staurenghi G, Arend O, Goger DG, Weiter JJ. In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics. Invest Ophthalmol Vis Sci. 1995;36:718-729.
3. Spaide RF. Fundus autofluorescence and age-related macular degeneration. Ophthalmology. 2003;110:392-399.
4. Keilhauer CN, Delori FC. Near-infrared autofluorescence imaging of the fundus: visualization of ocular melanin. Invest Ophthalmol Vis Sci. 2006;47:3556-3564.
5. Weinberger AW, Lappas A, Kirschkamp T, et al. Fundus near infrared fluorescence correlates with fundus near infrared reflectance. Invest Ophthalmol Vis Sci. 2006;47:3098-3108.
6. Sparrow JR, Boulton M. RPE lipofuscin and its role in retinal pathobiology. Exp Eye Res. 2005;80:595-606.
7. Holz FG, Schmitz-Valckenberg S, Spaide RF, Bird AC. Atlas of Fundus Autofluorescence Imaging. Berlin, Germany: Springer; 2007.
8. Jorzik JJ, Bindewald A, Dithmar S, Holz FG. Digital simultaneous fluorescein and indocyanine green angiography, autofluorescence, and red-free imaging with a solid-state laser-based confocal scanning laser ophthalmoscope. Retina. 2005;25:405-416.
9. Poloschek CM, Hansen LL, Bach M. Annular fundus autofluorescence abnormality in a case of macular dystrophy. Doc Ophthalmol. 2008;116:91-95.
10. von Ruckmann A, Fitzke FW, Bird AC. In vivo fundus autofluorescence in macular dystrophies. Arch Ophthalmol. 1997;115:609-615.
11. Helb HM, Charbel Issa P, van der Vehn RL, Berendschot TT, Scholl HP, Holz FG. Abnormal Macular Pigment Distribution in Type 2 Idiopathic Macular Telangiectasia. Retina. 2008;[in press].
12. Lois N, Halfyard AS, Bird AC, Holder GE, Fitzke FW. Fundus autofluorescence in Stargardt macular dystrophy-fundus flavimaculatus. Am J Ophthalmol. 2004;138:55-63.
13. Bindewald A, Bird AC, Dandekar SS, et al. Classification of fundus autofluorescence patterns in early age-related macular disease. Invest Ophthalmol Vis Sci. 2005;46:3309-3314.
14. Bindewald A, Schmitz-Valckenberg S, Jorzik JJ, et al. Classification of abnormal fundus autofluorescence patterns in the junctional zone of geographic atrophy in patients with age related macular degeneration. Br J Ophthalmol. 2005;89:874-878.
15. Bergmann M, Schutt F, Holz FG, Kopitz J. Inhibition of the ATP-driven proton pump in RPE lysosomes by the major lipofuscin fluorophore A2-E may contribute to the pathogenesis of age-related macular degeneration. Faseb J. 2004;18:562-564.
16. Dandekar SS, Jenkins SA, Peto T, et al. Autofluorescence imaging of choroidal neovascularization due to age-related macular degeneration. Arch Ophthalmol. 2005;123:1507-1513.
17. Holz FG, Bellman C, Staudt S, Schutt F, Volcker HE. Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration. Invest Ophthalmol Vis Sci. 2001;42:1051-1056.
18. Holz FG, Bindewald-Wittich A, Fleckenstein M, Dreyhaupt J, Scholl H, S. S-V. Progression of geographic atrophy and impact of fundus autofluorescence patterns in age-related macular degeneration. Am J Ophthalmol. 2007;143:463-472.
19. Schmitz-Valckenberg S, Bindewald-Wittich A, Dolar-Szczasny J, et al. Correlation between the area of increased autofluorescence surrounding geographic atrophy and disease progression in patients with AMD. Invest Ophthalmol Vis Sci. 2006;47:2648-2654.
20. Vaclavik V, Vujosevic S, Dandekar SS, Bunce C, Peto T, Bird AC. Autofluorescence imaging in age-related macular degeneration complicated by choroidal neovascularization: a prospective study. Ophthalmology. 2007;115:342-346.
21. Deckert A, Schmitz-Valckenberg S, Jorzik J, Bindewald A, Holz FG, Mansmann U. Automated analysis of digital fundus autofluorescence images of geographic atrophy in advanced age-related macular degeneration using confocal scanning laser ophthalmoscopy (cSLO). BMC Ophthalmol. 2005;5:8.
22. Schmitz-Valckenberg S, Jorzik J, Unnebrink K, Holz FG. Analysis of digital scanning laser ophthalmoscopy fundus autofluorescence images of geographic atrophy in advanced age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol. 2002;240:73-78.
23. Schmitz-Valckenberg S, Bultmann S, Dreyhaupt J, Bindewald A, Holz FG, Rohrschneider K. Fundus autofluorescence and fundus perimetry in the junctional zone of geographic atrophy in patients with age-related macular degeneration. Invest Ophthalmol Vis Sci. 2004;45:4470-4476.
24. Scholl HP, Bellmann C, Dandekar SS, Bird AC, Fitzke FW. Photopic and scotopic fine matrix mapping of retinal areas of increased fundus autofluorescence in patients with age-related maculopathy. Invest Ophthalmol Vis Sci. 2004;45:574-583.
25. Robson AG, Michaelides M, Saihan Z, et al. Functional characteristics of patients with retinal dystrophy that manifest abnormal parafoveal annuli of high density fundus autofluorescence; a review and update. Doc Ophthalmol. 2008.116:79-89.
26. Robson AG, Saihan Z, Jenkins SA, et al. Functional characterization and serial imaging of abnormal fundus autofluorescence in patients with retinitis pigmentosa and normal visual acuity. Br J Ophthalmol. 2006;90:472-479.
27. Radu RA, Han Y, Bui TV, et al. Reductions in serum vitamin A arrest accumulation of toxic retinal fluorophores: a potential therapy for treatment of lipofuscin-based retinal diseases. Invest Ophthalmol Vis Sci. 2005;46:4393-4401.
28. Bellmann C, Neveu MM, Scholl HP, et al. Localized retinal electrophysiological and fundus autofluorescence imaging abnormalities in maternal inherited diabetes and deafness. Invest Ophthalmol Vis Sci. 2004;45:2355-2360.