Fundus autofluorescence (AF) is a rapid, noncontact, noninvasive way to visualize fluorophores in the fundus. In this method, excitation light is introduced to the eye; fluorescence from intrinsic fluorophores is detected by using a barrier filter to exclude that excitation light from the image. The creation of fluorophores starts with visual pigment, which contains many conjugated double bonds. The visual pigment’s absorption of light energy may also lead to the creation of reactive molecular species that can cross-react to other molecules; 1 such molecule is the bis-retinoid A2E, a component derived from 2 molecules of vitamin A aldehyde and 1 molecule of ethanolamine. A2E appears to accumulate in Stargardt disease, in which a defective adenosine triphosphate–binding cassette protein (encoded by ABCA4) prevents proper transport of vitamin A derivatives across disc membranes in photoreceptors. This causes abnormal accumulation of 11-trans-retinal and several downstream reaction byproducts that are difficult for retinal pigment epithelium (RPE) cells to process. These products then accumulate in lysosomes as lipofuscin. Similar bis-retinoids accumulate within the lysosomes of RPE cells as a normal part of the aging process; they are not necessarily harmful. If the RPE cell dies, the contained lipofuscin disperses, resulting in a loss of autofluorescence, so that these areas appear dark on autofluorescence images. If there is a tear in the RPE, which is usually seen patients with choroidal neovascularization (CNV), the scrolled RPE is hyperautofluorescent because of reduplication, while the bared area shows no autofluorescence signal (Fig 2-6). This mechanism of autofluorescence loss is used to monitor the absence of RPE cells in a variety of diseases.
Figure 2-4 OCT angiography with projection artifact removal. A, Image showing a superficial vascular plexus with fractal branching. B, Image showing a deep vascular plexus. Its vessels are small and do not show the same branching characteristic as the superficial vascular plexus. C, Image of the choriocapillaris with dark areas; these low-signal areas are called signal voids.
(Courtesy of Richard F. Spaide, MD.)
Figure 2-5 Volume-rendered image of type 2 macular telangiectasia. The vessels at the level of the inner plexus are blue, those at the level of the deep plexus are red, and the vessels deep within the deep plexus, at the outer nuclear layer or below, are yellow. Note the tractional displacement of the vessels that leads to a near obliteration of the foveal avascular zone (asterisk). The foveal cavitations are shown in cyan.
(Courtesy of Richard F. Spaide, MD.)
There are 2 main types of systems used to image autofluorescence. A fundus camera uses special filters that are tuned to detect autofluorescence from lipofuscin without being swamped by autofluorescence from the crystalline lens, which may be derived, in part, from tryptophan and by nonenzymatic glycosylation of lens proteins. These cameras use wavelengths in the green end of the spectrum, and the recorded autofluorescence begins closer to the orange wavelengths. The excitation wavelengths are not absorbed by macular pigment. Commercial SLOs initially used blue excitation light intended to excite fluorescein, but these wavelengths were absorbed by macular pigment. Later, using a green laser for excitation was introduced; green light is not absorbed by macular pigment. By comparing the ratio of green light autofluorescence in 2 registered fundus images, it is possible to make a 2-dimensional map of macular pigment density.
Figure 2-6 Tears in the retinal pigment epithelium (RPE). A, Fundus photograph of a patient with choroidal neovascularization (CNV) who was given an intravitreal injection of an anti–vascular endothelial growth factor agent and developed what appeared to be 4 RPE tears (arrows).B, Fundus AF reveals the absence of autofluorescence and an increased signal where the RPE appears to be scrolled, thus confirming the CNRPE tear (arrows). Small areas of atrophy are also revealed by the autofluorescence (arrowheads).
(Courtesy of Richard F. Spaide, MD.)
Although lipofuscin in the RPE is the main source of autofluorescence from the fundus, accumulation of fluorophore in the subretinal space is another important signal source for the evaluation of some diseases, such as central serous chorioretinopathy. After the disease is present for a few months, the detachment becomes lighter and slightly more yellow in color, and the detachment also becomes hyperautofluorescent. This hyperautofluorescence is easier to detect with a fundus camera than with an SLO system for 2 main reasons. First, SLO systems are confocal; if the plane of focus is at the level of the RPE, the top of the detachment may not be in the confocal range. Second, the wavelengths used for fundus camera autofluorescence are more closely tuned to the fluorescence wavelengths emitted by fluorophores in the retina. Upon OCT imaging, an accumulation of material was found on the back surface of the retina in eyes with central serous chorioretinopathy with hyperautofluorescent detachments. It is thought that the photoreceptor outer segments are typically phagocytized and processed by the RPE, but if the retina has been physically elevated by fluid, the photoreceptors become separated from the RPE, thus impeding phagocytosis. This mechanism of disease pathophysiology may also be seen in vitelliform deposits in vitelliform macular dystrophy, adult vitelliform lesions, the yellow material that builds up under chronic retinal detachments caused by optic pit maculopathy, and pockets of retained subretinal fluid after detachment surgery.
Near-infrared fundus autofluorescence (NIA) imaging using 787-nm excitation and greater than 800-nm emission reveals fluorescence that was previously attributed to melanin from the RPE and the deeper layers of the choroid. However, lipofuscin can also fluoresce in the wavelengths mentioned, and it appears that during lipofuscin processing, melanosomes are fused with lysosomes to produce melanolysosomes. The melanin may bind to some of the free radicals in lipofuscin, but in any case, molecular cross-linking occurs. The resulting melanolipofuscin also fluoresces in the selected ranges. Thus, it may be possible to detect differing molecular species with autofluorescence. In addition, in time-resolved fluorescence imaging, different molecules can fluoresce at the same wavelengths but at measurably different times after excitation. Using time-resolved imaging with a phasor approach allows in vivo identification of various molecular species, as well as measurement of the reduction–oxidation reaction state. Studies are being conducted on hyperspectral autofluorescence in which differing wavelengths of fluorescence are measured in order to try to understand more about the formation of component molecules.
Schmitz-Valckenberg S, Holz FG, Bird AC, Spaide RF. Fundus autofluorescence imaging: review and perspectives. Retina. 2008;28(3):385–409.
Excerpted from BCSC 2020-2021 series: Section 10 - Glaucoma. For more information and to purchase the entire series, please visit https://www.aao.org/bcsc.