Fluorescence

Fluorescence is luminescence that is maintained only by continuous excitation. In other words, excitation at one wavelength occurs and is emitted immediately through a longer wavelength. Emission stops at once when the excitation stops. Fluorescence thus does not have an afterglow. A typical example of fluorescence is television. In the television tube, the excitation radiation is the electron beam from the cathode ray tube. This beam excites the phosphors of the screen, which re-emit the beam as a glow that constitutes a television picture.

Sodium fluorescein is a hydrocarbon that responds to light energy between 465 and 490 nm and fluoresces at a wavelength of 520–530 nm. The excitation wavelength, the type that is absorbed and changed, is blue; the resultant fluorescence, or emitted wavelength, is green–yellow. If blue light between 465 and 490 nm is directed to unbound sodium fluorescein, it emits a light that appears green–yellow (520–530 nm).

This is a fundamental principle of fluorescein angiography. In the procedure, the patient, whose eyes have been dilated, is seated behind the fundus camera, on which a blue filter has been placed in front of the flash. Fluorescein is then injected intravenously. Eighty percent of the fluorescein becomes bound to protein and is not available for fluorescence, but 20% remains free in the bloodstream and is available for fluorescence. The blue flash of the fundus camera excites the unbound fluorescein within the blood vessels or the fluorescein that has leaked out of the blood vessels. The blue filter shields out (reflects or absorbs) all other light and allows through only the blue excitation light. The blue light then changes those structures in the eye containing fluorescein to green–yellow light at 520–530 nm. In addition, blue light is reflected off the fundus structures that do not contain fluorescein. The blue reflected light and the green–yellow fluorescent light are directed back toward the film of the fundus camera. Just in front of the film a filter is placed that allows the green–yellow fluorescent light through but keeps out the blue reflected light. Therefore the only light that penetrates the filter is true fluorescent light (Fig. 1.1).

Pseudofluorescence

Pseudofluorescence occurs when nonfluorescent light passes through the entire filter system. If green–yellow light penetrates the original blue filter, it will pass through the entire system. If blue light reflected from nonfluorescent fundus structures penetrates the green–yellow filter, pseudofluorescence occurs (Fig. 1.2). Pseudofluorescence (i.e., fake fluorescence) causes nonfluorescent structures to appear fluorescent. It can confuse the physician interpreting the fluorescein angiogram and lead him or her to think that certain fundus structures or materials are fluorescing when they are not. Pseudofluorescence also causes decreased contrast, as well as decreased resolution. Because fluorescein angiography uses black-and-white film, the nonfluorescent or pseudofluorescent light appears as a background illumination. The background illumination from pseudofluorescence is especially heightened if there are white areas of the fundus, such as highly reflective, hard exudates. Pseudofluorescence must be avoided. Therefore the excitation (blue) and barrier (green–yellow) filters should be carefully matched so that the overlap of light between them is minimal.

Film-based versus digital fluorescein angiography – historical perspectives

Fluorescein angiography finds its origins in the late 1960s with the publication of an original article describing its use as well as subsequent atlases and textbooks for a medical retinal specialty in its infancy.1,6 The landmark text Atlas of Macular Diseases by Dr J. Donald Gass set a new standard for the use of stereoscopic fluorescein angiography in fundus diagnosis.⁷

As digital photography has evolved with improved resolution, the convenience of digital-based fluorescein angiography has gained wider acceptance. Though film-based images offer the highest amounts of resolution and 35-mm negatives are often easier to view for stereo, images are relatively difficult to manipulate, and training and effort are required to process and duplicate film. Transmitting or sharing film-based images is also time-consuming compared with digital images (Box 1.1).⁸

Camera and auxiliary equipment

Cameras differ in the degree of fundus area included in the photographs. Fundus cameras may range from 35° to 200° wide-field camera systems such as Optos.9,10 In clinical retinal practice, cameras ranging from 35° to 50° are routinely used (Fig. 1.3). Regardless of range, a camera with the ability to yield high resolutions of the posterior pole is essential for most macular problems, especially when laser treatment is to be done, as with background diabetic retinopathy, branch-vein occlusion, or choroidal neovascularization.

Wide-angle angiography has the benefit of capturing a single image of the retina in high resolution well beyond the equator. The potential for clinical efficiency and sensitivity in detecting neovascularization in the far periphery as well as acquiring an excellent clinical picture of the degree of capillary retinal nonperfusion is an exciting development in fluorescein angiography (Fig. 1.4).

Fluorescein solution

Sodium fluorescein, an orange–red crystalline hydrocarbon (C₂₀H₁₂O₅Na), has a low molecular weight (376.27 Da) and readily diffuses through most of the body fluids and through the choriocapillaris, but it does not diffuse through the retinal vascular endothelium or the pigment epithelium.

Solutions containing 500 mg fluorescein are available in vials of 10 mL of 5% fluorescein or 5 mL of 10% fluorescein. Also available are 3 mL of 25% fluorescein solution (750 mg). The greater the volume, the longer the injection time will be; the smaller the volume, the more likely a significant percentage of fluorescein will remain in the venous dead space between the arm and the heart (see Injecting the fluorescein, below). For this reason we prefer 5 mL of 10% solution (500 mg fluorescein).

Fluorescein is eliminated by the liver and kidneys within 24 hours, although traces may be found in the body for up to a week after injection. Retention may increase if renal function is impaired. The skin has a yellowish tinge for a few hours after injection, and the urine has a characteristic yellow–orange color for most of the first day after injection.

Various side-effects and complications can occur with fluorescein injection (Box 1.2).^11–15

A serious complication of the injection is extravasation of the fluorescein under the skin. This can be extremely painful and may result in a number of uncomfortable symptoms. Necrosis and sloughing of the skin may occur, although this is extremely rare. Superficial phlebitis also has been noted. A subcutaneous granuloma has occurred in a few patients after fluorescein extravasation. In each instance, however, the granuloma has been small, cosmetically invisible, and painless. Toxic neuritis caused by infiltration of extravasated fluorescein along a nerve in the antecubital area can result in considerable pain for up to a few hours. The application of an ice pack at the site of extravasation may help relieve pain. For extremely painful reactions an injection of a local anesthetic at the site of extravasation is effective but rarely necessary.

If extravasation occurs, the physician must decide whether to continue angiography. Extravasation may occur immediately, and thus the serum concentration of the fluorescein will be insufficient for angiography. In this case it usually is best to place the needle in another vein and reinject a full dose of fluorescein, starting the process again from the beginning. Occasionally, only a small amount of fluorescein is extravasated at the end of the injection. In this case photography can continue without stopping or reinjecting.

A common cause of extravasation is the use of a large, long needle directly attached to a syringe. It is difficult to hold the syringe in the dark. For this and other reasons we have discussed earlier, a scalp-vein needle attached to a syringe by a flexible tube is the best choice for this procedure. Also, the patient’s own blood can be drawn back into the tubing of the scalp-vein needle, with the blood going all the way up to but not into the syringe. When it is time to inject, the person giving the injection can look at the tip of the needle to ensure that extravasation has not occurred. If it has, the patient’s own blood is extravasated, and little chance of complication exists if the injection is stopped at this point so that no fluorescein is injected.

It is always important to watch for extravasation at the beginning of the injection so that, should it occur, the process can be halted; thus only a minimal amount of fluorescein will have been injected and extravasated. The amount of extravasated fluorescein can be minimized by slow injection and constant observation of the needle with a hand-held light or if injection is done before turning off the room lights.

Nausea is the most frequent side-effect of fluorescein injection, occurring in about 5% of patients. It is most likely to occur in patients under 50 years of age or when fluorescein is injected rapidly. When nausea occurs, it usually begins approximately 30 seconds after injection, lasts for 2–3 minutes, and disappears slowly.

Vomiting occurs infrequently, affecting only 0.3–0.4% of patients.11,13 When it does occur, it usually begins 40–50 seconds after injection. By this time most of the initial-transit photographs of the angiogram will have been taken. A receptacle and tissues should be available in case vomiting does occur. When patients experience nausea or vomiting, they must be reassured that the unpleasant and uncomfortable feeling will subside rapidly. Photographs can be taken after the vomiting episode has passed. A slower, more gradual injection may help to prevent vomiting.

Patients who have previously experienced nausea or vomiting from fluorescein injection may be given an oral dose of 25–50 mg of promethazine hydrochloride (Phenergan) by mouth approximately 1 hour before injection. Promethazine has proved to be helpful in preventing or lessening the severity of nausea or vomiting. We have recently found that we can also reduce the incidence of nausea by warming the vial of fluorescein to body temperature and drawing it into the syringe through a needle with a Millipore filter. Restriction of food and water for 4 hours before the fluorescein injection may reduce the incidence of vomiting; an empty stomach may prevent vomiting but will not affect nausea. If the patient still has a tendency to vomit despite taking all these measures, a lesser amount of fluorescein can be given and injected more slowly if the photographic results will not be compromised.

Vasovagal attacks occur much less frequently during fluorescein angiography than nausea and are probably caused more by patient anxiety than by the actual injection of fluorescein. We have seen vasovagal attacks even when the patient sees the needle or immediately after the skin has been penetrated by the needle but before the injection has begun. Occasionally a vasovagal reaction causes a patient to faint, but consciousness is regained within a few minutes. If early symptoms of a vasovagal episode are noted, smelling salts usually reverse the reaction. The photographer must be alert for signs of fainting because the patient could be injured if he or she falls.

Shock and syncope (more severe vasovagal reaction) consist of bradycardia, hypotension, reduced cardiovascular perfusion, sweating, and the sense of feeling cold. If the photographer and person injecting see that the patient is getting “shocky” or lightheaded, the patient should be allowed to bend over or lie down with their feet elevated. The patient’s blood pressure and pulse should be carefully monitored. It is important to differentiate this from anaphylaxis, in which hypotension, tachycardia, bronchospasm, hives, and itching occur.

Hives and itching are the most frequent allergic reactions, occurring 2–15 minutes after fluorescein injection. Although hives usually disappear within a few hours, an antihistamine, such as diphenhydramine hydrochloride (Benadryl), may be administered intravenously for an immediate response. Bronchospasm and even anaphylaxis are other reactions that have been reported, but these are extremely rare. Epinephrine, systemic steroids, aminophylline, and pressor agents should be available to treat bronchospasm or any other allergic or anaphylactic reactions. Other equipment that should be readily available in the event of a severe vasovagal or anaphylactic reaction includes oxygen, a sphygmomanometer, a stethoscope, and a device to provide an airway. The skilled photographer observes each patient carefully and is alert to any scratching, wheezing, or difficulty in breathing that the patient may have after injection.

There are a few published and unpublished reports of death following intravenous fluorescein injection. The mechanism may be a severe allergic reaction or a hypotensive episode induced by a vasovagal reaction in a patient with pre-existing cardiac or cerebral vascular disease. The cause of death in each case may have been coincidental. Acute pulmonary edema following fluorescein injection has also been reported.

There are no known contraindications to fluorescein injections in patients with a history of heart disease, cardiac arrhythmia, or cardiac pacemakers. Although there have been no reports of fetal complications from fluorescein injection during pregnancy, it is current practice to avoid angiography in women who are pregnant, especially those in the first trimester.

Aligning camera and photographing

To align the fundus camera properly, the photographer must first assess the “field of the eye.” The camera is equipped with a joystick with which the photographer can adjust the camera laterally and for depth. The camera is also equipped with a knob for vertical adjustment. The photographer finds the red fundus reflex, which is an even, round, sharply defined, pink or red light reflex. If the camera is too close to the eye, a bright, crescent-shaped light reflex appears at the edge of the viewing screen or a bright spot appears at its center. If the camera is too far away, a hazy, poorly contrasted photograph results.

The photographer moves the camera from side to side to ascertain the width of the pupil and the focusing peculiarities of the particular cornea and lens. The photographer studies the eye through the camera lens, moving the camera back and forth and up and down, looking for fundus details (e.g., retinal blood vessels). The photographer then determines the single best position from which to photograph (Figs 1.6 and 1.7).

Occasionally, a patient has a peculiar corneal reflex or central lens opacity, and it may be impossible to follow the usual procedure of aligning the camera through the central axis of the eye. Moving the camera slightly off axis may help improve focus and resolution.

Any abnormalities, such as an unusual light reflex or a poorly resolved image that the photographer sees through the camera system, will appear on the photograph. If the ophthalmoscopic view seen through the camera is not optimal, the photograph will not be optimal (Fig. 1.8). If the view is optimal, well aligned, in focus, and without reflexes, the photograph can be optimal. A helpful concept for the photographer is “what you see is what you get (or worse – never better).”

Focusing

Achieving perfect focus is a major factor in the photographic process. Both the eyepiece crosshairs and the fundus details must be in sharp focus to obtain a well-resolved photograph. The proper position of the eyepiece is determined by the refractive error of the photographer and the degree to which he or she accommodates while focusing the camera.

The photographer first turns the eyepiece counterclockwise (toward the plus, or hyperopic, range) to relax his or her own accommodation; this causes the crosshairs to blur. The photographer then turns the eyepiece slowly clockwise to bring the crosshairs into sharp focus. The eyepiece is focused properly when the crosshairs appear sharp and clear (Fig. 1.9). They must remain perfectly clear while the photographer focuses on the fundus with the camera’s focusing detail. With experience, the photographer becomes expert in adjusting the eyepiece and in keeping the crosshairs in focus throughout the entire photographic sequence.

The best position for the eyepiece is the point at which the crosshairs are in focus while the photographer’s accommodation is relaxed. Photographers learn to relax accommodation by keeping both eyes open. The photographer focuses the eyepiece with one eye and, with the other eye, keeps a distant object, such as the eye chart, in sharp focus. This skill may be difficult for technicians without ophthalmic training, but it is seldom impossible to learn.

Keeping the crosshairs in sharp focus, the photographer then turns the focusing dial on the camera to focus the fundus detail. Some photographers focus the crosshairs just once at the beginning of each day and control their accommodation throughout the day. This is not a good idea because the photographer’s accommodation may change during a photographic session; the photographer should be aware of this possibility and regularly check and readjust the eyepiece for focus. With the camera properly aligned and focused, the photographer is ready to start the preliminary photographs and angiograms.

Photographing the periphery

Photographing the peripheral retina with a standard 50° fundus camera demands precision and skills acquired only after many hours of practice. Problems with patient position and camera alignment and focus are compounded by marginal corneal astigmatism, unsteadiness of patient fixation, light reflexes, and awkward camera placement. All steps necessary for taking posterior photographs, such as alignment and focusing, must be employed to achieve good peripheral fundus photography. The Zeiss camera comes with an astigmatic dial to help neutralize the induced astigmatism. A tilt mechanism, now standard on most cameras, helps position the camera for extreme superior and inferior peripheral photography (Fig. 1.11).

During photography of the periphery, the patient tends to turn or move his or her head. Unsatisfactory photographs caused by the movement of the head away from the camera or to the side can be avoided if the photographer is alert to these possibilities and takes the necessary steps to prevent them. On the whole, achieving good peripheral photographs depends on photographic skill, of course, but also on patience on the part of both photographer and patient.

Informing the patient

An important step toward a successful angiogram is to inform the patient about the procedure. An informed patient is generally less anxious and more cooperative than one who is unsure of the situation. Some institutions routinely provide a consent form to be signed by patients who are to have angiograms. However, this practice cannot replace the duty of the physician to inform the patient about the procedure and its potential complications and to answer all questions.

The patient should be told that the eyes will be dilated, sodium fluorescein will be injected in a vein in the arm or back of the hand, and photographs will be taken. The patient should be assured that the flash is a harmless, bright light (not an X-ray) and that fluorescein dye is safe. The patient should be told that injection of the dye can cause complications but that such occurrences are rare. If the patient requests further details about complications, the physician is obligated to supply the information.

Positioning the patient

Before the patient is seated at the camera, the photographer makes sure that the front lens is free of any dirt or dust. The lens should always be covered by a lens cap when the camera is not in use. The front of the lens should be kept clean using chloroform and a tightly rolled rod of lens tissue. To clean the lens, begin at the center and rotate out to the periphery.

The patient is positioned at the camera with the chin in the chinrest and the forehead against the head bar. Because the most common cause of poor fluorescein photographs is involuntary movement of the patient’s head, the photographer should prepare and make adjustment for this before the fluorescein is injected. The photographer should aim and focus the camera on the specific area of primary interest, at the same time noting the patient’s responses. If the photographer finds that the camera must continually be moved closer to the patient while aligning it or taking preliminary photographs, or if reflexes suddenly appear in the view even though the camera is steady, then the patient’s head has moved away from the chinrest. If so, the photographer can make some adjustment before injecting the fluorescein dye. Sometimes having an assistant hold the patient’s head in the chinrest is helpful (Fig. 1.12). The photographer either may lower the entire camera and chinrest or raise the patient’s chair. This causes the patient to lean forward in the chinrest and against the forehead bar, making it more difficult for the patient to pull back.

Before photography begins, and between shots, the photographer may ask the patient to blink several times. This usually makes the patient more comfortable and also moistens the cornea and keeps it clear. When the pictures are actually being shot, the patient should be instructed to blink as infrequently as possible.

The photographer should talk to the patient frequently during the procedure, informing the patient of the progress of the testing and assuring him or her that all is going well. Explanation and reinforcement help produce better photographs.

Injecting the fluorescein

The color stereoscopic fundus photographs are taken first, before the fluorescein is injected. For injection, we recommend a syringe with a 23-gauge scalp-vein needle (Fig. 1.13). The scalp-vein needle has several advantages: it is small enough to enter most visible veins, and an intravenous opening is then available in the event of an emergency. Once in the vein, it requires no further attention, and although it can be taped in place, this usually is unnecessary. Whenever an antecubital vein is not visible or accessible, the vein in the back of the hand or radial (thumb) side of the wrist can usually be used for injection. Injecting the fluorescein into a hand or wrist vein increases the circulation time by a few seconds, but this seldom makes any difference.

Injection of the fluorescein is coordinated with the photographic process and is done after the first photographs (color fundus and control photographs; see next section) have been taken. With the needle in place, angiography can begin. By a predetermined, preferably silent, signal (such as a nod of the head), the photographer indicates to the physician to begin injecting fluorescein. The photographer starts the timer on the camera simultaneously with the start of injection and takes one photograph. This frame will show zero time on the photograph. In this way, the time from the beginning of injection is recorded on each subsequent angiographic photograph. When the injection is finished, the photographer may take another picture, which shows how long the injection took.

A rapid injection of 2 or 3 seconds delivers a high concentration of fluorescein to the bloodstream for a short time and probably yields somewhat better photographs than does a slower injection. However, the more rapid the injection, the greater the incidence of nausea from a highly concentrated bolus of fluorescein. For this reason a slower injection (4–6 seconds) is preferable; the photographs will still be of good quality. Because some fluorescein dye remains in the tubing, the scalp-vein needle should have short, rather than long, tubing to ensure that more of the dye is injected.

Developing a photographic plan

To photograph and print the fluorescein angiogram, we suggest the following comprehensive plan, designed to yield maximal angiographic information from each fundus and to facilitate a thorough and complete interpretation (Fig. 1.14). In contrast to film-based angiograms which required multiple duplicate attempts to assure at least one image would be optimal, fewer photographs are necessary in digital angiography. Although most angiograms will be complete by following this procedure, there will be exceptions. This plan must be modified if abnormalities occur in areas other than the macula and disc.

The photographic strategy essentially begins when the clinician has identified a condition or finding that requires angiographic study. The pathology dictates whether the photographic approach should image a magnified highly detailed finding versus a wider field of view for a more diffuse disease. Narrower field limits with higher magnification yield optimal images for focal pathology in conditions such as maculopathies, optic nerve disorders, and small focal lesions. Wider field of view often sacrifices magnification, but is effective in documenting conditions involving the periphery, such as diabetic retinopathy and vascular occlusive disease. Peripheral retinal scans for areas of neovascularization, as well as consideration for images that can be later montaged for wide-field reproductions, must also be incorporated into certain photographic plans. Elevated lesions such as tumors require great care in capturing high-quality stereo images.

It is both essential and extremely cost-effective for the physician to indicate specifically what areas to photograph. The photographer should be directed as to where to start the angiogram and the issues important for each specific angiogram. It is most efficient to use a photographic instruction slip that indicates the specific number of color photographs to take of each area, where to start the angiogram, what the diagnosis is, and any other information about the patient or fundus that is pertinent to the photographic process (Fig. 1.15). Although digital color and angiograms avoid the issue of wasted film and developing costs, unnecessary computer storage of images and patient inconvenience can be avoided with good technique and a repeatable, accurate algorithm for angiography.

Historically, because the roll of 35-mm negative film used for fluorescein angiography has 36 frames, it was convenient to think of the photograph session in terms of six rows of six frames each. Thus frame 1 appears in the upper right-hand corner and frame 36 in the lower left-hand corner. The angiogram developed from 36-mm negatives thus reads from right to left and from top to bottom. With the advent of digital imaging, theoretically, an unlimited number of frames can be acquired. However, to maximize efficiency of resources, digital storage of 20 frames per digital proof sheet is typically more than adequate for most clinical scenarios.

The first frame of the angiographic series is the color photograph of each eye. Then, a preinjection “control” photograph checks the dual-filter system for autofluorescence and pseudofluorescence.

At this point the fluorescein injection is begun. The needle is inserted in a vein in the patient’s arm (Fig. 1.16). The photographer waits for confirmation of successful venous access and awaits verbal confirmation that infusion is about to begin. Once the injecting clinician starts the infusion of fluorescein, the photographer begins the initial “injection” image. When the injecting clinician has completed infusion, he or she announces “injection complete” and the photographer takes the “end-of-injection” image. Because it is important to observe the site of the needle tip for extravasation of fluorescein, the lights are turned off only at the end of the injection. An alternative method is to turn the lights off after the needle has been inserted in the vein. The person injecting can hold a hand light to observe the fluorescein flow into the vein to be sure extravasation is not occurring. With the lights off, the photographer can become dark-adapted, which allows him or her to be better able to see the flow of fluorescein into the fundus as it occurs.

So as not to miss the appearance of fluorescein as it enters the fundus, the photographer should begin taking the initial-transit fluorescein photographs 8 seconds after the beginning of the injection of the dye if the patient is young and 12 seconds after injection for older patients. This is done so that these early photographs will not miss the appearance of fluorescein as it enters the fundus. Then, at intervals of 1.5–2 seconds, approximately 6 photographs should be taken in succession.

If the photographer does not see fluorescein entering and filling the retinal vessels while the six initial-transit photographs are taken, he or she must continue to photograph the fundus until filling takes place and also should check to see why no fluorescein is present.

After the first six initial-transit photographs and approximately 20–30 seconds after injection, with sufficient fluorescein concentration in the eye, the photographer should take a photograph of the fellow eye, a stereoscopic pair of photographs of the primary area of interest, followed by a stereoscopic pair of other pertinent areas. For example, in the suggested photographic plan, after stereophotographs of the right macula are taken, stereophotographs of the right disc are taken. The photographer should then photograph in stereo the macula and disc of the fellow eye.

Late-stage angiographs, preferably in stereo, are taken of the pertinent areas of each eye. It is important to photograph both discs and macula and any other areas of abnormal fluorescence and to note any areas that could not be photographed. This ensures that the interpreter will have adequate information for a complete interpretation of the angiogram.

The entire photographic process lasts 5–10 minutes. Angiophotographs taken more than 10 minutes after injection are usually not necessary. In some cases of central serous chorioretinopathy, or other rare situations, angiophotographs taken longer than 10 minutes after injection are helpful. This photographic algorithm is modified for specific conditions. For instance, in an angiogram of a diabetic patient, peripheral scans may be included at the request of the clinician surveying for neovascularization. In a patient with possible choroidal neovascularization due to age-related macular degeneration, additional angiophotographs of the suspicious lesion may be useful.

At the end of the session the patient is asked regarding any sensations related to an allergic reaction and reminded that the urine will be discolored for about a day.

In the event of a technical difficulty, such as camera breakdown, repeat fluorescein injection or photography can be carried out with satisfactory results after a waiting period of 30–60 minutes.

The plan we have suggested allows the fluorescein angiogram to yield all the information necessary to make a proper and thorough interpretation.

Box 1.3 provides a checklist of important steps in the fluorescein angiography procedure.

Fundus anatomy and histology

Fluorescein angiography has greatly increased our knowledge of retinal and choroidal circulatory physiology and fundus pathology. This clinical and research tool facilitates the in vivo study of histopathologic characteristics of fundus disease. Before the advent of fluorescein angiography, conditions such as pigment epithelial detachment, cystoid retinal edema, and subretinal neovascularization could be evaluated and understood only histologically. Now they are widely appreciated and recognized clinically. Because fluorescein angiography graphically demonstrates fundus pathophysiology, and because we rely on histologic points of reference to interpret a fluorescein angiogram, a thorough knowledge of the anatomy of the fundus and its microscopic layers is necessary to interpret fluorescein angiograms correctly. To interpret a fluorescein angiogram, it is essential to understand the microscopic layers of the fundus (i.e., the histology).

A logical place to begin this study is at the vitreous. In its normal state, and in a normal angiogram, the vitreous is clear and nonfluorescent. However, when it contains opacities that block the view of retinal and choroidal fluorescence, hypofluorescence occurs. The vitreous is also an important point of reference when intraocular inflammation or retinal neovascularization is present. In these cases fluorescein leaks into the vitreous, causing fluffy fluorescence as fluorescein molecules disperse into fluid vitreous and vitreous gel.

For the purpose of fluorescein angiographic interpretation, it is convenient to divide the sensory retina into two layers: the inner vascular half and the outer half, which is avascular. The inner vascular half extends from the internal limiting membrane to the inner nuclear layer. This portion of the retina contains the retinal blood vessels, which are located in two separate planes: the larger retinal arteries and veins are located in the nerve fiber layer; the retinal capillaries are located in the inner nuclear layer. In a well-focused stereoscopic fluorescein angiogram, these two vascular layers can be seen as distinct planes in the retina. An extremely important fluorescein angiographic concept is that normal retinal blood vessels are impermeable to fluorescein leakage; that is, fluorescein flows through the normal retinal vessels without leakage into the retina.

The outer avascular half of the sensory retina comprises the outer plexiform layer, the outer nuclear layer, and the rods and cones. The outer plexiform layer is the primary interstitial space in the retina. When the retina becomes edematous, it is in this layer that fluid accumulates, causing cystoid spaces. Deep retinal hemorrhages and exudates (lipid deposits) may also be deposited in the outer plexiform layer.

The rods and cones are very loosely attached to the pigment epithelium, especially in the macular region, whereas the pigment epithelium is very firmly attached to Bruch’s membrane. In fluorescein angiographic interpretation the pigment epithelium is an extremely important tissue because it prevents fluorescein leakage from the choroid and blocks, to a greater or lesser extent, visualization of choroidal fluorescence.

Bruch’s membrane separates the pigment epithelium from the choriocapillaris, which is permeable to fluorescein. Fluorescein passes freely from the choriocapillaris and diffuses through Bruch’s membrane up to, but not into, the pigment epithelium. Beneath the choriocapillaris are the larger choroidal vessels, which are impermeable to fluorescein. Melanocytes are dispersed throughout the choroid but are most heavily concentrated in the lamina fusca, the thin layer between the choroid and sclera. The sclera lies beneath the choroidal vessels.

The ophthalmic artery gives rise usually to two main posterior ciliary arteries: the lateral and medial. However, three posterior ciliary arteries may be present, in which case the medial artery is the one usually duplicated less frequently. In rare instances there may be a superior posterior ciliary artery.

The posterior ciliary arteries supply the lateral and medial halves of the disc and choroid. During angiography a vertical zone of slightly delayed filling may be seen passing through the papillomacular region, including the disc. Occasionally, there is an oblique orientation to this supply or even a superoinferior distribution. This border between the main posterior ciliary arteries has been termed the watershed zone, where patchy choroidal filling often can be seen on fluorescein angiograms.

Each main posterior ciliary artery divides into numerous short arteries and one long artery. On the temporal side the short posterior ciliary arteries supply small, variously sized, wedge-shaped choroidal segments, whose apices are centered near the macula. The lateral long posterior ciliary artery passes obliquely through the sclera. It supplies a wedge of choroid that begins temporal to the macular region and participates in the formation of the greater circle of the iris.

The choriocapillaris is made up of discrete units called lobules, thought to be approximately one-fourth to one-half of a disc diameter in size. The center of each lobule is fed by a precapillary arteriole (terminal choroidal arteriole), which comes from a short posterior ciliary artery. Each lobule functions independently in the normal state. It has been assumed that angiographic zones of delayed or patchy choroidal filling gradually fill in a transverse fashion, with one lobule spilling over into another. Careful inspection, however, indicates that these filling defects generally remain the same size, indicating a delayed filling from a posterior origin (its own arteriolar feeder). In the abnormal state, as when a choroidal vascular occlusion occurs, there is a freely connecting “spilling over” of blood flow from well-perfused choroid to the occluded area.

Around the margin of each lobule is a ring of postcapillary venules that drain each lobule. These postcapillary venules drain into the vortex veins, which drain the entire choroid. There are usually four vortex veins, and each functions as a well-defined quadrantic segmental drainage system for the entire uvea. In the case of a posterior ciliary artery obstruction, this occluded portion of the choroid can fill by a retrograde mechanism from an adjacent posterior ciliary artery by way of the choroidal venous system. This mechanism may provide adequate nourishment to prevent extensive ischemic changes until the occluded artery reopens.

Knowledge of each of these layers of the fundus is important in understanding fundus histopathology. The following six areas, however, are more important than others in the interpretation of abnormal fundus fluorescence:

Throughout this chapter a modified schematic drawing relates various fluorescein angiographic abnormalities to fundus histopathologic changes (Fig. 1.17). The size and proportion of these various layers have been modified to include various pathologic manifestations and to illustrate the effects of these abnormalities on the angiogram. Because of its importance and various pathologic changes, the pigment epithelium is drawn to a larger scale in relation to other fundus structures. Only the inner portion of the sclera is represented because the outer portion of the sclera is usually of little importance to angiographic interpretation. The retinal and choroidal vessels are drawn larger and more numerous than they appear in a normal histopathologic section to emphasize the contribution of circulatory pathophysiologic interpretation.

Two specialized areas of the fundus warrant more detailed discussion: the macula (Fig. 1.18) and the optic nerve head. The fovea is the center of the macula and contains only four layers of the retina: (1) the internal limiting membrane; (2) the outer plexiform layer; (3) the outer nuclear layer; and (4) the rods and cones. No intermediate layers exist between the internal limiting membrane and the outer plexiform layer in the fovea, which in the macula is oblique. This is an important factor in understanding the stellate appearance of cystoid edema in the macula as opposed to the honeycomb appearance of cystoid edema outside the macula. Beyond the macular region the outer plexiform layer is perpendicular rather than oblique.

The pigment epithelial cells in the macula are more columnar and have a greater concentration of melanin and lipofuscin granules than in the remainder of the fundus.

Xanthophyll is present in the fovea, located probably in the outer plexiform layer. These differences in pigmentation are the chief factors responsible for producing the characteristic dark zone in the macular region on normal angiograms. The absence of retinal vessels in the fovea (i.e., the perifoveal capillary-free zone), in most cases approximately 400–500 mm in diameter in the center of the fovea, is another cause of the dark appearance of the macula.

The optic nerve head, or disc, is the other highly specialized tissue of the posterior pole. The disc is fed by two circulatory systems: the retinal vascular system and the posterior ciliary vascular system. Widespread anastomotic channels exist between the posterior ciliary vasculature and the optic nerve and retinal vasculature and become exaggerated in certain pathologic conditions. The disc is made up of many layers of nerve fibers and glial supporting columns that contain the large retinal vessels.

The central retinal artery arises from the ophthalmic artery in close proximity to the main posterior ciliary arteries. In about 45% of the population, the central retinal artery and the medial posterior ciliary artery arise from a common trunk. In 12% of persons the central retinal artery originates from the ciliary artery. Therefore it is impossible to have a choroidal infarction, anterior ischemic optic neuropathy, and a central retinal artery occlusion all due to a single site of obstruction.

The central retinal artery provides a major source of blood supply to the axial portion of the anterior orbital portion of the optic nerve. In the intraneural or axial course, short centrifugal branches arise but usually end a short distance behind the lamina cribrosa. There are then no further branches from the central retinal artery until it reaches the retina. If a cilioretinal artery is present, it supplies the corresponding segment of the disc.

The peripapillary nerve fiber layer is supplied by small, recurrent branches from the retinal arterioles at the peripapillary region. Emanating from these arterioles at the disc are the radial papillary capillaries. These capillaries are rather straight and long, have few anastomoses, and lie in the superficial portion of the peripapillary nerve fiber layer. The capillaries to the disc are continuous with these retinal peripapillary capillaries.

The short posterior ciliary arteries, or the recurrent branches from the peripapillary choroid, supply the retrolaminar portion of the optic nerve. The laminar cribrosa portion of the nerve is supplied by centripetal branches of the short posterior ciliary arteries. In this region a partial, or, rarely, a complete Zinn’s vascular circle is occasionally found. The prelaminar portion is supplied by centripetal branches from the peripapillary choroid.

Because most of the disc is fed by the ciliary system, fluorescein appears simultaneously at the optic nerve head and the choroid and before it is apparent in the retinal arteries.

The main venous drainage of the disc is into the central retinal vein. The prelaminar portion empties into both the central retinal vein and the peripapillary choroid, thus providing potential collateral drainage in the case of obstruction of the central retinal vein behind the lamina cribrosa. Such large dilated collaterals are frequently seen following central retinal vein occlusion and are called retinociliary veins. Some mistakenly call them opticociliary shunts, a misnomer because they are not true shunts (defined as a congenital artery that empties into a vein and that skips the capillary bed, sometimes part of the Wyburn–Mason syndrome), and they are not optico because they emanate from the retina. They are, most accurately, retinovenous to ciliovenous collaterals.

In summary, fluorescein angiography provides an in vivo understanding of the histopathologic and pathophysiologic changes of various fundus abnormalities. Therefore an anatomic and, more specifically, a histologic understanding of important fundus landmarks is essential to fluorescein angiographic interpretation.

Normal fluorescein angiogram

The normal fluorescein angiogram is distinguished by certain specific characteristics. Knowledge of these characteristics provides an essential frame of reference for interpreting abnormal fluorescein angiograms.

In the normal fluorescein angiogram (Fig. 1.19), the first true fluorescence begins to show in the choroid approximately 10–12 seconds after injection in young patients (e.g., adolescents) and 12–15 seconds after injection in older patients.

Fluorescence can appear even earlier than 8 seconds in very young patients. The choroid occasionally begins to fluoresce 1 or 2 seconds before the initial filling of the central retinal artery. Early choroidal fluorescence is faint, patchy, and irregularly scattered throughout the posterior fundus. It is interspersed with scattered islands of delayed fluorescein filling. This early phase is referred to as the choroidal flush. When adjacent areas of choroidal filling and nonfilling are quite distinct, the pattern is designated as patchy choroidal filling.

Within the next l0 seconds (approximately 20–25 seconds after injection), the angiogram becomes very bright for about 5 seconds because of the extreme choroidal fluorescence. Choroidal fluorescence, however, is not visible in the macula because of the taller, more pigmented epithelium present in the fovea (retina). Therefore the macula remains dark throughout the angiogram.

If present, a cilioretinal artery usually begins to fluoresce as the choroid fluoresces, rather than as the retina fluoresces. Within 1–3 seconds after choroidal fluorescence is visible, or approximately 10–15 seconds after injection, the central retinal artery begins to fluoresce. The less dense the concentration of pigment in the pigment epithelium, the greater the time between the visibility of the choroidal fluorescence and the filling of the retinal vessels. The lighter pigment presents less interference to choroidal fluorescence, allowing it to be evident earlier in its filling phase. With a more densely pigmented pigment epithelium, the blockage barrier effect is greater. Therefore choroidal fluorescence appears somewhat later because a greater concentration of fluorescein is required to overcome the increased density of the pigment epithelial barrier.

Because no barrier exists in front of the retinal vessels, the patient’s pigmentation has no effect on the visibility of the retinal vessels, although the degree of pigmentation does affect the contrast of the angiophotographs. The darker the pigment epithelium is, the less visible the choroidal fluorescence will be and the greater the contrast of the retinal vascular fluorescence (i.e., the better they stand out). The lighter the pigment epithelium is, the more visible the choroidal fluorescence will be and the less the contrast of the fluorescence from the retinal vessels.

After the central retinal artery begins to fill, the fluorescein flows into the retinal arteries, then into the precapillary arterioles, the capillaries, the postcapillary venules, and finally the retinal veins. Because the fluorescein from the venules enters the veins along their walls, the flow of fluorescein in the veins is laminar. Because vascular flow is faster in the center of a lumen (tube) than on the sides, the fluorescein seems to stick to the sides, creating the laminar pattern of retinal venous flow. The dark (nonfluorescent) central lamina is nonfluorescent blood that comes from the periphery, which takes longer to fluoresce because of its more distant location.

In the next 5–10 seconds, fluorescence of the two parallel laminae along the walls of the retinal veins becomes thicker. At the junction of two veins, the inner lamina of each vein may merge. This creates three laminae: one in the center and one on each side of the vein. As fluorescein filling increases in the veins, the laminae eventually enlarge and meet, resulting in complete fluorescence of the retinal veins.

Fluorescence of the disc emanates from the posterior ciliary vascular system, both from the edge of the disc and from the tissue between the center and the circumference of the disc. Filling also comes from the capillaries of the central retinal artery on the surface of the disc. Because healthy disc tissue contains many capillaries, the disc becomes fairly hyperfluorescent on the angiogram.

The perifoveal capillary net cannot always be seen on the fluorescein angiogram. It can be seen best in young patients with clear ocular media about 20–25 seconds after a rapid fluorescein injection. This is called the “peak” phase of the fluorescein angiogram. The photographer should be aware of this phase and be sure not to miss it by shooting as rapidly as possible as the fluorescein concentration increases and by continuing to shoot rapidly until the concentration of fluorescein begins to decrease.

Approximately 30 seconds after injection, the first high-concentration flush of fluorescein begins to empty from the choroidal and retinal circulations. Recirculation phases follow, during which fluorescein in a lower concentration continues to pass through the circulation of the fundus.

Generally, 3–5 minutes after injection, the choroidal and retinal vasculatures slowly begin to empty of fluorescein and become gray. Vessels of most normal patients almost completely empty of fluorescein in approximately 10 minutes. The large choroidal vessels and the retinal vessels do not leak fluorescein. However, because of large gaps in its endothelium, the choriocapillaris does leak fluorescein. The extravasated fluorescein diffuses through the choroidal tissue, Bruch’s membrane, and sclera. Leakage of fluorescein with retention in tissues is designated as staining. In the later phase of the angiogram, staining of Bruch’s membrane, the choroid, and especially the sclera may be visible if the pigment epithelium is lightly pigmented. The disc and adjacent visible sclera remain hyperfluorescent because of staining. When the retinal pigment epithelium is especially lightly pigmented, the large choroidal vessels can be seen in silhouette against the fluorescent (fluorescein-stained) sclera. The lamina cribrosa within the disc also remains hyperfluorescent because of staining. This depends on the cup-to-disc ratio and the presence of any visible sclera, such as occurs within a conus adjacent to the disc. The edge of the disc stains from the adjacent choriocapillaris, which normally leaks.

To summarize, the angiogram is initially dark; choroidal and retinal filling is seen 10–15 seconds after fluorescein injection. The retinal and choroidal vasculatures fill maximally about 20–30 seconds after injection. Late angiophotographs show fluorescence of the choroid and sclera (if the pigment epithelium is light) and fluorescence of the optic cup and the edge of the disc, but otherwise the fundus is dark (nonfluorescent in the late phase).

Hypofluorescence

Hypofluorescence is a reduction or absence of normal fluorescence, whereas hyperfluorescence is abnormally excessive fluorescence. A systematic series of decisions follows this initial differentiation to arrive at a proper diagnosis. These decisions relate to: (l) the anatomic location of various abnormalities; (2) the quality and quantity of the abnormal fluorescence; and (3) other unique characteristics, as indicated in Fig. 1.20.

Hypofluorescence is any abnormally dark area on the positive print of an angiogram. There are two possible causes of hypofluorescence: blocked fluorescence or a vascular filling defect.

Blocked fluorescence is sometimes referred to as masked, obscured, or negative fluorescence or transmission decrease. Each of these terms indicates a reduction or absence of normal retinal or choroidal fluorescence because of a tissue or fluid barrier located anterior to the respective retinal or choroidal circulation. For example, blood in the vitreous or a layer of blood in front of the retina obscures the view of the retinal and choroidal circulations and therefore blocks fundus fluorescence from these tissues. Hemorrhage that lies under the retina or retinal pigment epithelium, but in front of the choroidal circulation, does not obstruct visibility of the retinal circulation but does block the view of the choroidal circulation. Therefore the approximate histologic location of blocking material can be determined by the presence or absence of visibility of one or both fundus circulations.

Fluorescein is present but cannot be seen in blocked fluorescence. With vascular filling defects, however, fluorescein cannot be seen because it is not present.

The key to differentiating blocked fluorescence from a vascular filling defect is to correlate the hypofluorescence on the angiogram with the ophthalmoscopic view. If there is material visible ophthalmoscopically that corresponds in size, shape, and location to the hypofluorescence on the angiogram, then blocked fluorescence is present. If there is no corresponding material on the color photograph, then it must be assumed that fluorescein has not perfused the vessels and that the hypofluorescence is caused by a vascular filling defect.

Hypofluorescence resulting from a vascular filling defect occurs when either of the two fundus circulations is not perfusing normally. This is caused by an absence of the vascular tissue or by a complete or partial obstruction of the particular vessels. In these situations an absence or delay of fluorescence of the involved vessels will occur. This type of hypofluorescence has a pattern that follows the geographic distribution of the vessels involved. Although the ophthalmoscopic picture will demonstrate the material blocking fluorescence, it may show nothing if the hypofluorescence is the result of a vascular filling defect.

To summarize, after an area of hypofluorescence is recognized, one must refer to the ophthalmoscopic photograph to determine the cause. If material is visible ophthalmoscopically and corresponds to the area of hypofluorescence, this is blocked fluorescence. If no corresponding blocking material exists, the hypofluorescence is therefore a vascular filling defect.

Hyperfluorescence

Hyperfluorescence is any abnormally light area on the positive print of an angiogram, that is, an area showing fluorescence in excess of what would be expected on a normal angiogram. There are four possible causes of abnormal hyperfluorescence: (1) preinjection fluorescence; (2) transmitted fluorescence; (3) abnormal vessels; and (4) leakage. The appearance of fluorescence depends in part on the relationship of its appearance to the timing of the fluorescein injection.

Preinjection fluorescence is hyperfluorescence that can be seen before fluorescein is injected and is caused by structures that naturally fluoresce (autofluorescence) or by poorly matched filters (pseudofluorescence).

Transmitted fluorescence and abnormal vascular fluorescence occur in the early, or vascular, stage of the angiogram, when fluorescein fills patent blood vessels. Transmitted fluorescence appears when fluorescein fills the normal choriocapillaris, but it is more noticeable when there is reduced pigment in the pigment epithelium or loss of retinal pigment epithelium. This is designated pigment epithelial window defect.

When abnormal retinal, disc, or choroidal vessels are present and fill with fluorescein, hyperfluorescence occurs. This type of hyperfluorescence, abnormal vascular fluorescence, is also seen in the early, or vascular, phase of the angiography.

Hyperfluorescence caused by leakage is seen predominantly in the later, or extravascular, phase of angiography. In this phase, fluorescein has emptied from normal and abnormal vessels. Any significant fluorescein that remains in the eye is fluorescein that has escaped or leaked from vascular or tissue barriers and is thus extravascular.

Therefore, to ascertain the type of hyperfluorescence, one must determine the time at which the hyperfluorescence appears in relation to when the fluorescein was injected. Once the hyperfluorescence is determined to be caused by preinjection fluorescence, transmitted fluorescence, the presence of abnormal vessels, or by leakage, the next step is to determine the anatomic location of the hyperfluorescence. Abnormal blood vessels may come from the retina and disc or from the choroid. Leakage can occur in the vitreous, disc, retina, or choroid.

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