Figure 1. Normal retina. FAF Fundus Camera (Kowa VX-20; Designs For Vision)
BOptom PGDipAdvClinOptom PGCertOcTher FACO
Lead Optometrist Primary Care, Australian College of Optometry
Heidelberg Engineering BluePeak Blue Laser Autofluorescence
Canon CR-2 Plus AF
Zeiss Visucam 500
Fundus autofluorescence (FAF) is an imaging technique with broad applications in fundus examination and diagnosis of retinal disorders. New technology and research are driving a renewed interest in FAF and helping to establish it as another tool in the detection and treatment of ocular disease.
What is it?
When particular components of ocular tissues (fluorophores) are exposed to certain wavelengths of light, they can be coaxed to radiate a different spectrum of light in response. The emission of this light is called fluorescence. The term ‘autofluorescence’ is used to distinguish this ‘natural’ emission of light from fluorescence induced by the injection of dyes such as sodium fluorescein or indocyanine green into the eye.
The role of lipofuscin
The natural fluorophore in the retina is lipofuscin, accumulated in the retinal pigment epithelium (RPE), however, optic nerve drusen, astrocytic hamartomas and even the ageing crystalline lens can also autofluoresce. Lipofuscin excitation occurs with wavelengths between 300 and 600 nm, and autofluorescent emission is in the 480 to 800 nm range (maximum intensity at 600-640 nm).
Lipofuscin is a normal metabolic by-product of photoreceptor outer segment phagocytosis by the RPE. Its background concentration normally increases with age.
Excessive lipofuscin accumulation in the RPE may indicate RPE cell distress or an abnormal metabolic demand on the tissue. It can be used as a precise delineator of retinal dysfunction in many conditions including age-related macular degeneration, retinitis pigmentosa, vitelliform dystrophy and plaquenil maculopathy.
The intensity of fluorescence is dependent on the concentration of lipofuscin in the RPE. By visualising lipofuscin distribution within the RPE, fundus autoflorescence imaging enables observation of in situ retinal function, before more overt structural signs become evident on fundoscopy or optical coherence tomography (OCT).
Clinically, two varieties of instrument are available for FAF imaging: the confocal scanning laser ophthalmoscope cSLO (for example, Heidelberg BluePeak Blue Laser Autofluorescence) and retinal camera systems with FAF capability (for example, the Canon CR-2 Plus AF, the Zeiss Visucam 500, the Kowa VX-20 and the Topcon TRC-NW8F).
The cSLO system produces a FAF image by using a low-energy blue argon laser to excite the lipofuscin in the RPE, and a barrier filter to selectively capture only the lipofuscin response. A cSLO system, typically coupled with OCT, can perform multiple repeat scans, which are averaged to produce a single high-contrast high-resolution image. Confocal optics also allow the laser to better reach the retinal plane, bypassing possible interference from other source of ‘autofluorescent noise’ in the eye, such as cataract.
Digital funuds camera
FAF retinal cameras use a high-energy flash and a broadband Spaide exciter filter to penetrate the ocular media and excite the lipofuscin. The autofluorescence response is captured through a barrier filter for the entire field of capture. However, because no multiple-scan averaging is performed, the image often has less resolution compared to a cSLO device.
Because the fundus camera does not use a coherent (laser) beam to illuminate the retina, the lipofuscin response is weaker and more prone to scatter from opaque media than the cSLO FAF image.
Both FAF imaging systems require significant amounts of light to illuminate the retina, which may cause visual discomfort to patients. It is also worth noting that there is currently no standardisation of FAF acquisition and processing settings among different manufacturers.
The clinical role of FAF imaging
FAF images are monochromatic 256-greyscale, with dark areas representing regions of low lipofuscin (hypofluorescence) and bright areas of high lipofuscin (hyperfluorescence) (Figures 1 and 2). The posterior pole of a healthy fundus has an overall diffuse, mildly hyperfluorescent appearance due to the normal background lipofuscin levels in the RPE.
Figure 2. Normal retina. FAF image from cSLO system (Heidelberg Spectralis BluePeak Autofluorescence). Note the difference in capture field of view and resolution compared with Figure 1. The characteristic dark hypofluorescence is noticeable at the optic nerve, retinal blood vessels and fovea.
Optic nerve head, retinal blood vessels and fovea appear darker (hypofluorescent) against mild background hyperfluorescence, because of total absence of RPE; signal blocking by haemoglobin and macular carotenoid pigments, respectively.
Focal hypofluorescence represents loss or drop-out of RPE cells. Conversely, localised hyperfluorescence is indicative of increased lipofuscin content, and active degenerative or oxidative stress on the RPE or photoreceptors. For example, in atrophic macular degeneration, RPE integrity is disturbed, with both hypo-and hyperfluorescent areas present at the macula FAF image. (Figure 3A)
Figures 3A and 3B. Atrophic (dry) age-related macular degeneration. Note the patchy FAF pattern, with both hypo- and hyperfluorescent areas near the fovea (Heidelberg Spectralis BluePeak Autofluorescence). (Retinal image: Canon CR-1 digital camera)
By comparison, in disciform geographic atrophy, or similar end-stage irreversible RPE drop-out (pan-retinal photocoagulation, toxoplasmosis scarring, and so on) FAF will show circumscribed hypofluorescence due to total loss of RPE activity. (Figure 4A)
Figure 4A and 4B. Geographic atrophy (age-related macular degeneration). Note the pronounced circumscribed foveal hypofluorescence, corresponding to the area of RPE loss, and the ring of hyperfluorescence at the edge of hypofluorescence, suggesting the AMD condition is likely to extend and worsen. A further noteworthy feature is an area of reticular pseudodrusen supero-temporal to the macula. (Heidelberg Spectralis BluePeak Autofluorescence) (Retinal image: Canon CR-1 digital camera)
Some studies1 suggest that presence of hyperfluorescence at the edges of geographic atrophy may be indicative of a higher likelihood of progression, due to ongoing degenerative activity in the junctional RPE cells (Figure 4A). This is one example where longitudinal FAF imaging could allow more precise tracking of progression of AMD and possibly earlier detection of change than fundoscopy or OCT.
Similarly, lipofuscin tends to accumulate in many retinal and macular dystrophies (for example: Stargardt’s and Vitelliform) producing characteristic zones of hyperfluorescence, and providing clues for diagnosis and progression (Figures 5 and 6).
Figure 5. Adult vitelliform macular dystrophy. Note the characteristic highly fluorescent FAF pattern at the fovea, indicating excess lipofuscin accumulation in the RPE. A small area of hypofluorescence is present in the centre of the lesion, suggesting RPE and photoreceptor atrophy (Heidelberg Spectralis BluePeak Autofluorescence)
Figure 6. Retinal image showing the characteristic ‘yellow’ deposit at the macula (Canon CR-1 digital camera)
Fundus autofluorescence provides a non-invasive in situ method of visualising the functional integrity of the RPE; a means of monitoring lipofuscin accumulation and detection of subtle morphological changes in the macula pigment in the retina. FAF can be invaluable for retinal examination and in conjunction with other methods of fundus assessment (for example, fundoscopy, OCT, photography) can offer optometrists a new tool for the detection and monitoring of many retinal conditions.
1. Holz FG, Bindewald-Wittich A, Fleckenstein M et al; FAM-Study Group. Progression of geographic atrophy and impact of fundus autofluorescence patterns in age-related macular degeneration. Am J Ophthalmol 2007; 143: 3: 463-472.