FUNDUS AUTOFLUORESCENCE

1. FUNDUS AUTOFLUORESCENCE
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 Lipofuscin
• 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. Lipofuscin
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
Common Challenges –
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. Differences 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 Autofluorescence
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

28. 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

29. • 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 FA 36 sec FA 69 sec

30. Geographic Atrophy
⦁ 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.

31. ⦁ 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

32. 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.

33. Junctional zone activity classification

34. DIFFUSE

35. 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

36. 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).

37. 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).

38. 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

39. 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

40. (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.

41. 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.

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

43. 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.

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

45. 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.

46. 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

47. 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

48. 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.

49. 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

50. 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

51. 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

52.  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;

53.  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.

54. 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.

55. 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.

56. • 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

57. 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

58. 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

59. 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.

60. parafoveal
pattern
Normal
Fundus
pericentral
pattern
Mixed
pattern

61. 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

62. 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

63. Thank You