Fluorescence: the property of emitting a longer wavelength light when stimulated by a shorter wavelength

White light from the camera passes through a blue excitation filter
Blue light (wavelength of 490nm) is transmitted to the fundus and is absorbed by the fluorescein molecules in the retinal and choroidal vasculature
They are stimulated to emit yellow-green light (530nm)

Both the excitation and fluorescence of fluorescein angiography are absorbed and scattered by retinal pigments meaning that structures deep to these tissues are poorly visualised

Macular xanthophyll may have a physiological role in limiting damage to the photoreceptors by protecting them from high energy short-wavelength light

A yellow-green filter is placed to block blue light reflected from the eye
The yellow-green light therefore reached the camera
Fluorescein has a low molecular weight

Can pass between endothelial cells in the choriocapillaris and other capillary beds in the body (therefore, in other tissues it does not stay intravascular)
The RPE tight junctions normally prevent this leak into the neural retina (outer barrier)
The tight junctions between retinal vascular endothelial cells also prevent leak normally (inner barrier)
Fluorescein is 80-85% bound to serum protein (it is primarily hydrophilic)
Leaks readily from fenestrated choriocapillaris
10% concentration fluorescein is used

Anaphylactic shock: <1 in 100,000 (<0.001%)
More common side effects

Discolouration of skin and urine
Nausea and vomiting
Urticarial rash
Flushing
Photosensitivity
Itch
Discomfort at injection site

The diameter of retinal vessels on FFA is larger than that of a fundus photograph

A photo only visualises the axial blood column whereas fluorescein reaches the peripheral blood in the vessel

## Uses

Diagnosis

AMD
CSR
CMO
Diabetic retinopathy
Retinal vein occlusion
Posterior uveitis
Disc drusen
Choroidal haemangiomas

Management

To guide laser in CNV, CSR, diabetic maculopathy and vein occlusion

## Normal phases

Choroidal: 10-12 seconds after injection in young patients but depends on vascular health

A cilioretinal artery, if present, is a branch of the short posterior ciliary artery so fills during the choroidal phase

Arterial: 1-3 seconds after the choroidal phase

Neovascularisation of the disc (part of the retinal circulation) is visible during the early arterial phase
The central retinal artery provides a circuitous route for the dye so it appears 1 or 2 seconds later than in the choroidal circulation

Arteriovenous

Demonstrates laminar flow: the veins are fluorescent near their walls and darker centrally
Lasts for 1-2 seconds

Venous
Late phase

Highlights cystoid macular oedema, CSR, or occult subretinal neovascular membranes

Late staining of the optic disc is normal

## Abnormalities

Pooling: hyperfluorescence that increases in size and intensity as dye enters a closed space eg a pigment epithelial detachment

Once the space is filled, the hyperfluorescence remains stable (cp leakage)

Leakage: increases in size and intensity of hyperfluorescence over time (due to extravasation of dye)

Incompetence of the inner or outer blood-retinal barriers
Neovascularisation: defective inner barrier
Defective choroidal circulation eg. AMD

Window defect: unmasking of the normal choroidal fluorescence

RPE atrophy
Window defects are seen early

Staining: hyperfluorescence appears late. Uptake of dye into structures

Drusen
Disciform scars
Optic disc

Pseudo-autofluorescence: overlap in the spectral transmission of the excitation and barrier filters

## ICG

Provides more information about the choroidal circulation

ICG peak absorption (790-805nm) and fluorescence (770-880nm) are within the infrared range of wavelengths (>800nm) and thus can penetrate the overlying RPE, pigments and any overlying haemorrhages

ICG circulates 98% bound to plasma protein (it is amphiphilic, ie. both hydro- and lipophilic therefore binds lipproteins and phospholipids) so less leakage
Therefore provides information about the choroid that would be invisible to FFA due to extravasation of fluorescein from the choroid
Takes longer than FFA
Excreted by the hepatobiliary system: discoloured stool for several days
Useful for

Occult/poorly defined CNVM: can be more easily measured
Polypoidal CNV
Fibrovascular PEDs
Medial opacities/vitreous haemorrhages
Photophobic patients (they cannot see the infrared lights)
Inflammatory disease: to identify possibly occult choroidal disease (crucial investigation for inflammatory disease)

Summary Table: FFA findings of important pathologies table

Classic CNV	Well-delineated ‘lacy’ hyperfluorescence in early phasesAggressive/progressive leaking leads to dye pooling in subretinal space
Occult CNV	‘Stippled’ hyperfluorescence early with increasing pooling in the sub-RPE space (fibrovascular PED) Diffuse hyperfluorescence late with poorly defined boundaries (late leakage of indeterminate origin)
RAP	Early hyperfluorescence with progressive leakage
PCV	ICG better: shows early subretinal hyperfluorescence <5 minutes
CSR	Expansile dot (/ink-blot)Smoke-stack pattern
CMO	Parafoveal leakage into petaloid pattern (cystoid spaces)
Myopic CNV	Smaller than ARMD CNV with less subretinal/intraretinal fluid
Angioid streaks	Hyperfluorescence due to window defect
OIS	delayed/patchy choroidal filling, prolonged AV transit time, macular leakage/oedema, optic disc hyperfluorescence, retinal vascular staining (due to endothelial damage). Peripheral capillary non-perfusion
Coats	Highlights abnormal vessels with leakage and areas of capillary dropout
MacTel type 2	Telangiectatic capillaries temporal to the fovea with early leakage. Signs of CNV if present
Macroaneurysm	Immediate complete filling with late leakage
Stargardt’s	Dark choroid due to blockage of choroidal fluorescence by abnormal deposits in RPE (of lipofuscin and A2E)
Hydroxychloroquine toxicity	Windows defects due to RPE loss
Capillary haemangioma	Rapid filling of a feeding artery followed by complete hyperfluorescence of the lesion, then filling of the vein and diffuse late leakage
Cavernous haemangioma	Slow incomplete filling of lesion with fluid meniscus and no leakage
Racemose haemangioma	Rapid filling with no leak