reflection. The two are sometimes correlated, but there are structures, like melanin in the RPE or erythrocytes in blood vessels, which are capable of multiply reflecting near-infrared light, but because of these reflections, the coherency information is lost. In these situations, objects located behind scattering structures can look dark (are “shadowed”) even though the shadowing object did not absorb the light. Using OCT, the various layers of the retina can be differentiated. The true identity of these layers has not been definitively established for some of the layers. What has been called the outer nuclear layer, attributed to a hyporeflective layer in the outer retina, is probably made up of the outer nuclear layer and the outer part of the outer plexiform layer.

9.6Fundus Angiography

Fundus angiography is done with two different dyes, sodium fluorescein, which principally images the retinal circulation and indocyanine green, which is primarily used for evaluating the choroidal circulation [1]. Fluorescein angiography was developed first, is much more useful, and more is known about the correlation between histopathology and angiographic findings than for indocyanine green. At present there are only a few specific indications for indocyanine green angiography, and the significance of many of the indocyanine green angiographic findings is not known with certainty. Therefore, fluorescein angiography serves as the archetype for learning fundus angiography. However, indocyanine green angiography has offered new insights into the pathophysiology of different types of choroidal neovascularization (CNV), and serves as the chief method of diagnosing one particular subtype of CNV, namely polypoidal choroidal vasculopathy.

9.6.1Fluorescein Dye Characteristics

Fluorescein sodium has a molecular weight of 376.27 Dalton and it is highly water soluble. The dye is removed from the vascular compartment by the kidney, and patients with renal disease may not be able to adequately clear fluorescein. The fluorescein molecule is excited by light in the range of 465–490 nm, a bluish-green color, and enters into a higher energy state. The molecule emits a longer wavelength fluorescence, between 520 and 530 nm, a greenish-yellow color, as it decays to a lower energy state. The emitted fluorescence is linked tempo-

rally with the excitation, each quanta of stimulating light causes an almost immediate release of a quantum of fluorescent light. In a fundus camera, a filter, called an excitation filter, is placed over a broadband light source and allows the passage of the blue-green light. This light stimulates the fluorescein in the eye, which emits yel- low-green light. The light reaching the camera has both the yellow-green fluorescence and some reflected bluegreen excitation light. A barrier filter is used to block the reflected blue light, allowing only the fluoresced yellowgreen light to pass through. This resultant fluorescence is recorded by a detection system, which in the past was film but now in almost every instance employs a CCD camera. Scanning laser ophthalmoscopic systems use a laser light with a wavelength of 488 nm that is scanned across the fundus and a longpass filter starting at 500 nm is used to block the laser wavelengths.

The excitation and barrier filters are chosen to have very little overlap in the wavelengths transmitted to avoid any extraneous light bleeding through the filters to reach the detector. Some structures, such as hard exudates or optic nerve drusen, reflect a great amount of the excitation back to the camera. Because of a small overlap in filter systems used in fluorescein angiography, some of this reflected light may pass through the barrier filter and appear as a false fluorescence, or pseudofluorescence. This type of fluorescence results from a defect in the matching of the filter, and is largely avoided with the use of modern filters. Other materials deposited in the fundus, such as lipofuscin, fluoresce when exposed to the excitation light. This fluorescence will be recorded on the film prior to injection of dye and is called autofluorescence.

9.6.2Indocyanine Green Dye Characteristics

ICG is stimulated by the absorption of infrared light in the range from 790 to 805 nm. The dye emits fluorescent light between 825 and 835 nm. A barrier filter blocks reflected light shorter than 825 nm. The retinal pigment epithelium and the choroid absorb a much larger proportion of the light used for fluorescein angiography than the near-infrared light used for ICG angiography. Scattering is also wavelength dependent, with longer wavelength to be more extensive, than shorter wavelengths. As ICG absorbs and fluoresces in the nearinfrared spectrum, there is an enhancement of visualization, as compared with fluorescein angiography,

through the RPE, serosanguinous fluid, and shallow hemorrhages. Although the fluorescence efficiency of ICG seems much lower than that of sodium fluorescein, the higher transmittance of light above 800 nm and the strong intravascular retention of the ICG dye allow a better visualization of the choroidal vascular architecture than in fluorescein angiography. About 25% of the intravascular ICG is removed per minute by the liver. Patients with liver problems can have slow removal of the dye from their blood streams.

9.6.3Cameras Used in Fluorescence Angiography

The two principal camera designs are based on a scanning laser ophthalmoscope and on a fundus camera. Scanning laser ophthalmoscopes rely on focused laser light that is swept across the fundus in a raster pattern. Scanning laser ophthalmoscopes are capable of confocal imaging, where only light from a conjugate plane of interest is detected by the image sensor. This permits rejection of light from planes that are not of interest. This optical property is one factor that helps scanning laser ophthalmoscopes achieve increased contrast of vascular structures. A second strength of scanning laser systems is they record images in real time at a high frame rate. Accurate filling of different elements in the vascular tree leading to and coming from an area of neovascularization is possible. Selective laser targeting of these selected vessels forms the strategy behind feeder vessel ablation techniques in the treatment of choroidal neovascularization. Observing the motion of the dye front through the ocular vasculature allows the brain to integrate information over time, which improves the ability to perceive vessels.

The second method of recording images is to use a fundus camera–based system. These systems utilize a bank of capacitors that are discharged through a xenon flash tube. Resultant photographs are recorded as a single image, usually digitally. The frame rate of a fundus camera is limited by the time to recharge the capacitors, the speed at reading the CCD camera, and the time involved in storing the results in memory. Most fundus cameras cannot take more than 1 or 2 frames per second. Fundus cameras do not have confocal imaging, and therefore image every source of fluorescence in the optical pathway. On the other hand, fundus camera images have a greater pixel count with lower noise than scanning laser ophthalmoscopic pictures.

9.6.4Patient Consent and Instruction

The risks of fluorescein angiography should be outlined by the physician. Every patient has a yellowish appearance after fluorescein angiography. Their urine will be particularly yellow after the angiography. The most common side effects include nausea (about 5%) and vomiting, and the development of hives (also about 5%) [17, 18]. Patients with a past history of problems are much more likely to experience them with repeat fluorescein angiography [17]. The feeling of nausea passes in a few seconds without treatment. Hives, unless very mild, are usually treated with diphenhydramine or similar antihistamine. Extravasation of dye at the injection site is painful and causes a yellow spot that lasts for a day or so. More serious side effects such as hypotension, shock, laryngeal spasm, and even death have occurred, but fortunately are quite uncommon.

Side effects from indocyanine green angiography seem less common than with fluorescein angiography, possibly because indocyanine green has a higher amount of protein binding and may be less likely to stimulate the chemoreceptor trigger zone. Indocyanine green dye was first approved in 1956 for human use to study the hepatic and cardiac systems [19]. As a result, there are nearly 55 years of data related to the side effects available. Mild gastrointestinal disturbance, itching, or hives are uncommon with ICG [20–22]. Extravasation of injected ICG is painful and can cause a dark green spot that may last for several days. Overall, adverse reactions due to ICG occur less frequently than with fluorescein. The incidence of death after fluorescein injection is 1 in 222,000; for ICG, it has been estimated as 1 per 333,000 [18]. While ICG is safe in general use, it contains 5% iodine by weight. This is inorganic iodine, and the risk of administration in patients with allergies to organic iodine is not known.

On occasion patients who were allergic to shellfish were cautioned against iodine-containing radiocontrast agents, with little factual evidence to support the recommendation. The logical link between radiocontrast agents and ICG or between shellfish and the inorganic iodine in ICG preparations is even more tenuous as these patients still consume iodized salt. Patients with renal disease or with liver dysfunction may have a potentially increased risk to adverse reactions [23]. The dye has not been shown to cross the placenta, but no studies on fetal toxicity have been performed.

Both fluorescein and indocyanine green angiography are safe intraoffice procedures, but occasionally reactions,

Fig. 9.3 Early after injection (26 s) the retinal arterioles are filled with fluorescein dye and early filling of a choroidal neovascular membrane is seen (arrow). By 47 s after injection, the

retinal vessels are completely filled with fluorescein and leakage from the choroidal neovascularization is evident

sometimes severe, can occur. Patients must therefore be screened carefully for potential risk factors, and, as with all intravenously administered agents, adequate resuscitation facilities and properly trained personnel must be available to manage these problems. There is no agreement about the need of obtaining written consent for fundus angiography, but certainly, the patient should be made aware of the risks and benefits of angiography.

9.6.5Fluorescein Injection

The two most popular ways of injecting fluorescein is either as 5 ml of a 10% solution, or 2 ml of a 25% solution. There is not much functional difference between the two concentrations. The fluorescein to be injected is drawn into a syringe, and a 23-gauge butterfly is placed on the syringe. The butterfly is connected to a flexible clear tubing. This allows the person injecting the dye to be certain the needle is in the vein as the reflux of blood will be seen in the tubing. The antecubital vein is commonly used as it is large and readily accessible. In some patients, particularly if they are obese, the vessels on the back of the hand may be easier to see and inject. The disadvantages of the veins on the hand are the injection may be slightly more painful, the veins are more likely to roll, any ecchymosis after the injection is more likely to be visible, and the time for the dye to travel from the arm to the eye is sometimes used as a rough indication of ocular perfu-

sion, although it is influenced by numerous factors including location of speed of injection.

9.6.6Fluorescein Technique

After the injection, the photographer expectantly waits while looking at the fundus image. In young adults, the dye appears within 12 s; in older patents, the dye’s appearance may be delayed. At the first sign of fluorescence in the eye, a rapid series of photographs are taken (Fig. 9.3). With digital systems, it is common for the photographer to take many photographs early after injection because it is easy to delete unneeded frames. Fine adjustments on the focus or illumination are frequently required early in the angiographic series. After the early phase, the stereo-pairs can be taken of the macula and disk of the study eye as well of the fellow eye. Stereophotography was nearly universally done when film was used to record the image, but is less common with conventional digital imaging. There are stereoviewers available for digital angiography. If the patient has a macular problem, the photographer should visually scan the retina peripheral to the arcade with the camera, and should photograph any visible abnormality. Midphase photographs, taken 1–2 min after injection, are then completed. At about 5 or 6 min after the injection, additional stereo-pairs are taken of the macula and any pathology seen. Some clinicians also take a series of photographs at 10–15 min after fluorescein injection.