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

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