FUNDUS AUTOFLUORESCENCE

FUNDUS AUTOFLUORESCENCE

1. FUNDUS AU T OFLUO R ESCENCE Dr. Arindam Rakshit FNB Trainee Vitero-Retina Sir Ganga Ram Hospital, New Delhi

2. Definition It is a noninvasive imaging method for in vivo mapping of naturally or pathologically occurring fluorophores of the ocular fundus First described by Delori in the 1980s

3. Fluorophore absorbs photon of the excitation wavelength elevates electron to an excited, high energy state Dissipates energy through molecular collisions Emits a quantum of light at a lower energy and longer wavelength Ground State Basic Physics

4. Fundus Autofluorescence Major source of fundus autofluorescence is the lipofuscin of the RPE and melanin

5. RPE and L ipofuscin LF is a byproduct of accumulation of sheded outer segments of the photoreceptors Over years, each RPE cell will eventually phagocytize 3,000,000,000 outer segments Up to 25 percent of the cell volume will be occupied by lipofuscin

6. L ipofuscin WHY IS IT DANGEROUS ? A2-E (N-retinylidene-N-retinylethanol- amine) the dominant fluorophore possess toxic properties Interferes with the normal cell function Precursors of A2-E are also toxic Products of photo-oxidation of RPE lipofuscin serves as trigger for complement activation  inflammation

7. It absorbs blue light with a peak wavelength of 470 nm and emits yellow-green light at a peak wavelength 600-610 nm. Lipofuscin based autofluorescence is also known as blue autofluorescence (BAF), short wave autofluorescence (SWAF) or simply autofluorescence. 

8. Near-infrared autofluorescence (NIR-AF) Melanin has a peak excitation at wavelength of 787 nm and it emits fluorescence in the near-infrared region. It is distributed in the fovea, macula, and periphery. Melanin based autofluorescence is also known as near-infrared autofluorescence. (NIRAF)

9. NIR-AF images can be obtained by using ICGA mode of the scanning laser ophthalmoscope (SLO), i.e., without dye Due to the excitation and emission in the red end of the spectrum, so the LF is eliminated from the studied. NIR-AF signal is mainly melanin-derived and some contributions from choroidal sources.

10. Figure 1A: BAF image of normal left eye shows dark optic disc and blood vessels. The retina is greyish in color and the fovea (red ring) is dark as well. Figure 1B: NIRAF image of normal left eye shows dark optic disc and blood vessels. The retina is greyish in color and the fovea is (red ring) is the brightest point of the image. BAF and NIRAF

11. Red Free vs FAF

12. Effect of light exposure on autofluorescence imaging Light exposure changes the rhodopsin pigment present in the OS of rod photoreceptors. In a dark-adapted eye, rhodopsin remains active and absorbs excitation light leading to reduced autofluorescence signals. After prolonged exposure to light, it undergoes photo-isomerization and loses its absorption capacity. Hence prolonged light exposure increases the BAF signals to a significant level. This is called bleaching effect

13. Recording of Autofluorescence

14. The main barrier is the crystalline lens With development of nuclear lens opacities, the fluorescence of the lens becomes even more prominent. Therefore, fundus AF imaging with a conventional fundus camera using the excitation and emission filters as applied for FFA produces images with low contrast and high background noise .

15. Autofluorescence systems C ommon C hallenges – naturally occurring intrinsic fluorescence of the ocular fundus is quite low Confocal Scanning Laser Ophthalmoscope (cSLO) Modified Fundus Camera (mFC)

16. Scanning Laser Ophthalmoscopy cSLO addresses the limitations of the low intensity of the AF signal and the interference of the crystalline lens. It projects a low-power laser beam on the retina that is swept across the fundus in a raster pattern. The intensity of the reflected light at each point, after it passes through the confocal pinhole, is registered by means of a detector and a 2D image is subsequently generated. The use of confocal optics ensures that out-of-focus light is suppressed and thus the image contrast is enhanced. Excitation is induced in the blue range (λ = 488 nm), and an emission filter between 500 and 700 nm

17. Commonly used cSLO system Heidelberg retina angiograph/Heidelberg Spectralis . Rodenstock (no longer available) Zeiss prototype SM 30 (no longer available) Nidek F-10

18. Fundus Camera It shows a summation of fluorescence from the fundus and consequently can image fluorescence from the retina and RPE at the same time. It captures AF using a single flash of light. To reduce AF of the lens and cornea, filter is set with red-shifted wavelengths, with an excitation spectra of 535–585 and a 615–715 nm emission barrier filter

19. The use of the red-shifted wavelengths decreases absorption by macular pigments. Scattered light from structures outside the retinal plane may falsely increase the FAF signal, a phenomena termed pseudo-AF.

20. Optos ultra‑widefield system The Optomap Ultra-Widefield system by Optos combines confocal scanning laser technology with an ellipsoid mirror to achieve up to 200 degrees of view It simultaneously uses two excitation wavelengths of red (633  nm) and green (532  nm) light with an emission filter of >540 nm. The longer wavelength spectra of this system reduces absorption by macular pigment and allows for a clear image.

21. Advantage – The ability to acquire images through a native nondilated pupil, a brief image acquisition time (250  ms ), the option of pseudocolor fundus photography Disadvantage- are lid and eyelash artifact, lack of real-time averaging, and poor contrast

22. Range of excitation and emission for different camera systems. cSLO , confocal scanning laser ophthalmoscopy; FC , fundus camera

23. D ifferences between cSLO and mFC Confocal Scanning Laser Ophthalmoscope (cSLO) Modified Fundus Camera (mFC) One excitation wavelength (laser source) Large emission spectrum (cut-off filter) Bandwidths filters for excitation and emission Continuous scanning at low light in a raster pattern intensities One single flash at maximum intensities Confocal system Entire cone of light Laser power fixed by manufacture, gamma detector sensitivity Flash light intensity, and gain of detector adjustable. Automatic real time image processing with averaging of single frames and pixel normalization Manual contrast and brightness

24. CLINICAL APPLICATIONS

25. Normal Autofluoresc e nce distribution ⦿ Optic nerve head Absence of autofluorescent pigment ⦿ Retinal blood vessels Absorption by blood vessels ⦿ Foveal area Absorption by luteal pigment ⦿ Parafoveal area Mildly decreased intensity due to high melanin content and lower density of lipofuscin granules in central RPE

26. AF and ARMD- Basic Considerations RPE is thought to play a key role in the early and late phases of the disease Hallmark of aging is the accumulation of lipofuscin granules in the cytoplasm of the RPE cells Lipofuscin accumulation is the common downstream process

27. Ability to document spatial distribution of lipofuscin and its changes over time. The amount of autofluorescence is signature for previous or possible future oxidative injury Delori et. al mentioned that decreased FAF in the center of the druse with a surrounding annulus of increased FAF Hyperfluorescence in FAF F A 36 sec F A 69 sec

28. Geographic A trophy Representing the natural end-stage of AMD, when CNV does not develop . GA is characterised by the development of areas of outer retinal atrophy that slowly enlarge over time at a median rate of 1.5 to 2.1 mm2 per year.

29. Atrophic areas in GA lack RPE lipofuscin High-contrast difference between atrophic and non- atrophic retina allows the area of atrophy to be more precisely and accurate identified. Atrophic areas in GA can be detected by FAF as they appear as dark areas. These images represent the progression of GA, over time, in two patients

30. The striking finding of FAF imaging in GA patients is the frequent presence of areas of hyperautofluorescence in the junctional zone surrounding the patch of atrophy. Surrounding the atrophy, in the junctional zone, foci and areas of increased FAF intensity.

31. Junctional zone activity classification

32. DIFFUSE

33. FAM (Fundus Autofluorescence Imaging) study Aim of the study: to identify the rate of atrophy enlargement b/w patients. Which was neither explained by – the extent of baseline atrophy other comorbid factor like smoking, lens status, or family history

34. Result of the study: 195 eyes of 129 patients shows that variable rates of progression of GA are dependent on the specific phenotype of abnormal FAF pattern at baseline. The report indicates that eyes with the banded (median 1.81 mm2/year) and the diffuse FAF pattern (1.77 mm2/year) showed a more rapid enlargement of atrophy compared with eyes without FAF abnormalities (0.38 mm2/ year) and the focal FAF pattern (0.81 mm2/year).

35. AF in foveal sparing phenomenon “foveal sparing” progresses 2.8-fold faster towards the periphery than towards the central retina Detection of “foveal sparing” with BAF imaging is challenging due to signal absorption by macular pigment The approach combining infrared reflectance with FAF images can be identified the “foveal sparing” in eyes with GA. (A) Fundus autofluorescence imaging (excitation wavelength 488 nm); (B) near-infrared reflectance imaging; (C) spectral domain optical coherence tomography (vertical scan).

36. Choroidal neovascularization Early CNV is not readily detectable on FAF, reflecting intact RPE and photoreceptor layers Classic CNV appears hypo- autofluorescent due to blockage of the RPE by the type 2 fibrovascular complex in the subretinal space CNV may be bordered by hyper-autofluorescence in 38 % of cases due to associated RPE proliferation or photoreceptor loss resulting in a window defect

37. FAF patterns in dry AMD may predict the development of CNV Batoglu et al. found that the patchy, Linear and reticular patterns of early dry AMD had the strongest correlation with progression to neovascular AMD, with 30.4 % of eyes developing CNV the mean follow-up period of 29.2 months

38. (C,D) FFA reveals an active CNVM with leakage in the inferior part of the lesion. (B) On the AF image, the borders ( arrows ) of the subretinal fluid can be seen. Of note, the AF signal appears to be normal at the site of the active neovascularization, suggesting that the retinal pigment epithelium is still viable.

39. RPE tears RPE tears are a well-known complication of neovascular AMD, commonly fibrovascular PEDs > 450 TO 600 microns in height. It appear as a well-demarcated area of hypo-AF with adjacent hyper-AF in the form of rolled redundant RPE. Over time, tears remodel and resurfacing occurs, with recovery of autofluorescence extending centripetally from the borders toward the center.

40. The process of resurfacing correlates with visual improvement and may benefit from treatment with anti-VEGF, though studies differ

41. Central serous chorioretinopathy During the initial presentation of CSCR, 72–96  % of cases show hypo-AF corresponding to the focal leakage site on FFA and to the area of NSD, due to blockage by SRF. Localized PED shows focal hyper-AF As the disease progresses, there will be granular hyper-AF, with increased number and size of hyper-AF dots corresponding to subretinal precipitates on OCT. In chronic cases, 85  % showed hypo-AF corresponding to the atrophic gravitational tracts.

42. acute CSCR with hyper-AF material at the margin and inferior region hypo-AF gravitational tract from chronic inactive CSCR with hyper-AF margins

43. Autofluorescence in choroiditis In active stage there is diffuse hyper-AF of the entire lesion. As the lesion starts to heal with treatment it develops a rim of hypo-AF along with fading central hyper-AF. With complete healing of the lesion, the entire choroiditis area turns hypo-AF. Autofluorescence imaging may be a non-invasive tool to assess treatment response in choroiditis.

44. Color fundus photo before treatment - diffuse hyper-AF of entire lesion After treatment – A rim of hypo-AF with fading central hyper-AF Healed Stage – Diffuse hypo-AF

45. Macular Telangiectasia Reduced macular pigment density in MacTel type 2 affects this masking. They will show an abnormally increased signal in the macular area to blue-light FAF imaging Initially occur in the area temporal to the foveal center

46. Classification by Gass and Blodi class 1 shows a wedge-shaped loss of macular pigment restricted to an area temporal to the foveal center. class 2, the area is larger and involves the foveal center. Class 3 is characterized by loss of luteal pigment within anoval - shaped area centered on the foveola.

47. FFA (left) and FAF image of type 2 idiopathic macular telangiectasia showing an abnormal FAF distribution in the macular area due to depletion of luteal pigment

48. Retinitis pigmentosa FAF acts as a modality to monitor RP and correlate phenotype with genotype. Murakami et al. identified three subsets of RP on FAF 59 % hyper-AF parafoveal ring not visible on funduscopic exam, 18 % abnormal central hyper-autofluorescence extending centrifugally from the fovea, 24 % had neither pattern

49. Robson-holder ring FAF of retinitis pigmentosa shows an area of normal preserved retina at the posterior fundus bordered by a hyper-AF Robson-holder ring. Mottled hypo-autofluorescence outside the ring represents photoreceptor degeneration. These details are visible on FAF but not on fundus photography It corresponds to the border of IS/OS disruption

50. OCT analysis shows complete photoreceptor loss outside of the ring, with the ELM in direct apposition to the RPE. The ring itself corresponds to OS dysgenesis and lipofuscin production, while normal retina lies within the ring. The retinal sensitivity as measured by multifocal ERG correlates with the radius of the AF ring, indicating intact retinal sensitivity inside the ring but none outside. The size of the ring correlates with visual function as measured by perimetry;

51. More the ring encroached centrally, the more constricted the visual field Serial imaging may be helpful in determining the disease progression. Similar rings are also seen in other retinal dystrophies, Leber congenital amaurosis (LCA), bull’s eye maculopathy, X-linked retinoschisis, Best macular dystrophy, cone dystrophy, and cone-rod dystrophy This phenotype on FAF suggests an underlying common mechanism for the pathogenesis of retinal dystrophies.

52. Choroideremia Night blindness with centripetal atrophy of the choroid, RPE, and photoreceptor layer, though the macula is spared. FAF shows bilateral, symmetric, midperipheral zones of hypo-AF due to RPE atrophy. It will have scalloped edges with a preserved area of central stellate AF. FAF of asymptomatic female carriers shows a peripheral speckled pattern of hyper-AF In conjunction with genetic testing, FAF is a useful for evaluating female relatives of affected patients.

53. Stargardt disease Most common hereditary juvenile macular dystrophy Results from an autosomal recessive mutation in the ABCA4 gene. There is defective OS degradation, lipofuscin accumulation, and central degeneration of the RPE and photoreceptor layer. Clinically, there is foveal atrophy surrounded by yellow flecks, peripapillary sparing, and central vision loss.

54. Areas of atrophy on fundus corresponded to hypo- AF Flecks seen as depigmented lesion appeared as areas of hypo- autofl Predictive value is yet to be determined

55. Macular dystrophies: Best’s disease Vitelliform stage : central round area of increased FAF Pseudohypopyon stage: increased FAF in the lower part Late stages: irregular FAF within the lesion with disseminated spots of increased FAF

56. Macular dystrophies: Best’s disease Pattern of spread on FAF: centrifugal Atrophic regions are associated with low levels of background FAF, lower visual acuity, abnormal colour vision, central scotomas and poorer electrophysiological results FAF appears more striking and widespread

57. Drug toxicity Hydroxychloroquine toxicity The risk for ocular toxicity rises after cumulative dose over 1000g There is irreversible parafoveal photoreceptor loss with foveal sparing. FAF shows a hyper-AF parafoveal ring corresponding to photoreceptor damage. Later there will be hypo-florescence due to RPE atrophy.

58. parafoveal pattern Normal Fundus pericentral pattern Mixed pattern

59. Screening Guidelines According to AAO annual examinations starting at 1 year of use and yearly evaluation with diagnostic testing including SDOCT, perimetry, and mf-ERG Compared to multi-focal ERG, FAF has a sensitivity of 73.7 % It is best used as a component of multi-modal imaging in the screening process

60. Deferoxamine‑induced retinal toxicity It is characterized by pigmentary changes with RPE mottling, vitelliform lesions, and bull’s eye maculopathy. Viola et al. conducted case control study of β-thalassemia who received deferoxamine, and developed FAF abnormalities. They proposed the below four distinct FAF patterns: minimal change, focal, patchy, speckled patterns Patients with the patchy or speckled pattern had the most severe vision loss

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