The instrumentation used for fluorescein angiography must be capable of delivering the proper excitation wavelengths and capturing rapid sequence images of the retina as the dye courses through the vasculature. The modern mydriatic fundus camera is the tool most commonly used for this purpose, but scanning laser ophthalmoscopes and some specialized wide-field fundus cameras can also be used. Fluorescein angiography can be performed using 35mm black-and-white panchromatic films or with digital cameras. A number of major ophthalmic instrument manufacturers produce fluorescein-ready fundus cameras in both film and digital configurations. Third-party vendors offer digital conversion solutions for a variety of film-only cameras.
Although fluorescein angiography could be done in color, black-and-white imaging offers increased light sensitivity and ease of contrast enhancement to compensate for the low levels of fluorescence in the bloodstream. Film-based angiography requires either the use of a processing service, or access to a darkroom for processing films on-site. Films with an ISO film speed rating of 400 or higher are used and push processed in high contrast developers to increase both light sensitivity and contrast. Films developed in this way exhibit an increase in the silver halide grain structure and a reduction in apparent resolution. These films however are often more forgiving of exposure inconsistencies than are digital cameras
The fundus camera is a horizontally mounted instrument with an internal electronic flash and an attached 35mm SLR or digital sensor. Successful fundus imaging relies on the interaction between the optics of the fundus camera with the optics of the eye itself. Fundus cameras utilize an aspheric design, that when combined with the optics of the subject eye, matches the plane of focus to the curvature of the fundus. Although it is essentially a low-power microscope placed just an inch or two from the subject, proper focus is set at or near infinity, because when aimed into the eye, the light path exiting the fundus camera then passes through the refracting optics of the cornea and the natural lens which are usually focused at distance upon dilation. The focus control of the fundus camera is used to compensate for refractive errors in the subject eye. Conditions such as myopia or astigmatism are routinely encountered and many fundus cameras have additional controls to compensate for these optical imperfections in patients’ eyes.
The light needed to illuminate the fundus is delivered axially. The optical system of the fundus camera projects a ring of light from the internal strobe through the dilated pupil. The ring shape allows a separation of the outgoing and incoming illumination. The ring fills the outer pupil with light that reflects off the retina, exits the pupil through the center of the ring, and continues through the optics of the fundus camera to form an image of the fundus at the film plane.
Fundus cameras are often described by their optical angle of view. An angle of 30 degrees is considered the normal angle of view and creates a film image 2.5 times life size. Fixed-angle cameras usually offer the sharpest optics, but variable-angle cameras provide wide-angle capabilities between 45 and 60 degrees. Wide-angle cameras need to illuminate a broader area of retina, requiring a more-widely dilated pupil to accommodate a larger ring of light.
Fundus cameras equipped for fluorescein angiography have a timer that records the angiographic sequencing on each frame of the study, a matched pair of exciter and barrier filters and a fast recycling electronic flash tube that allows a capture rate of up to 1 frame per second. Narrow band-pass interference filters are utilized to allow maximum transmission of peak wavelengths, while minimizing any crossover of transmission curves. The exciter filter transmits blue-green light at 465-490 nm, the peak excitation range of fluorescein. The barrier filter transmits a narrow band of yellow at fluorescein’s peak emission range of 520-530 nm. The barrier filter effectively blocks all visible wavelengths but the specific color of fluorescein.
On rare occasions, fluorescein angiography of the iris or other anterior structures may be of value to ophthalmologists. This can be accomplished by using the plus-diopter setting on the fundus camera, but images may be slightly distorted. Alternately, fast-recycling power packs and matched filter sets can be adapted to a photo slit-lamp to achieve high-quality results.
The ophthalmic community was quick to adopt digital imaging technology for fluorescein angiography. Commercial digital systems designed specifically for fluorescein angiography and retrofitted to existing film-capable fundus cameras began to appear on the market as early as 1983.1 This configuration continues to be the most common type of instrumentation used for angiography, offering choice and flexibility between film and digital camera backs. In recent years, new instruments have been developed that rely entirely on digital capture technology for angiography. Digital-only devices include scanning laser ophthalmoscopes and specialized wide-field retinal imaging devices that can be configured for fluorescein angiography. Digital imaging hardware and software continues to evolve and improve in quality, and we are likely to see more digital-only devices for angiography in the future.
Spatial resolution in current retinal imaging systems varies from 800 x 600 to over 3000 x 2000 pixels. Monochrome digital backs are considered better for angiography than their color counterparts since they are usually more light sensitive, and all pixels are available for exposure to fluorescence. The narrow band of wavelengths generated by fluorescein will expose only the green channel pixels in RGB color sensors, reducing resolution through interpolation. However, both monochrome and color CCDs with pixel counts of 3 Megapixels or above surpass the spatial resolution of the 400 ISO films commonly employed for film-based angiography. Resolution comes at a cost in terms of light efficiency. High-resolution digital sensors require more light for proper exposure and flash settings often need to be increased. New high-transmission filter sets have been developed to improve light efficiency and performance with high-resolution digital sensors.
Digital imaging offers several distinct advantages over traditional film-based angiography. Computer technology offers a variety of powerful tools that can be used to enhance diagnostic information. Software tools provide adjustments for brightness, contrast and sharpness. Digital analysis enables measurement of pathologic structures, digital overlays can be used to identify potential changes in lesion size in serial photographs, and multiple fields can be linked together to form composite wide-field images. Images can be stored on optical media like CD or DVD and transmitted electronically across computer networks for remote viewing or storage on servers.
Commercially available digital angiography systems typically conform to the Digital Imaging and Communications in Medicine (DICOM) Standards. This is a set of universal standards for transferring diagnostic images and associated information between devices manufactured by different vendors.2-5 Conformance to these standards facilitates connectivity to picture archiving and communication systems (PACS) used in radiology, and integration with electronic medical records and other hospital information systems.
In addition to well-known advantages in capturing, processing, enhancing, storing and distributing images electronically, having instant access to digital images can increase clinical efficiency and enhance patient education opportunities through the ability to review images on large screen computer monitors with the patient. Another significant advantage to digital imaging is that it can shorten the learning curve for novice angiographers trying to master the complex techniques of angiography. Having instant feedback allows the operator to adjust exposure settings and camera alignment to correct any flaws in technique.
Stereo imaging techniques can be used during angiography to enhance diagnostic information. Stereo images provide a visual sense of depth that is particularly useful in identifying the histopathologic location of angiographic findings within the retina. Stereo separation is achieved by laterally shifting the fundus camera a few millimeters between sequential photographs. The lateral shift causes the illuminating beam of the fundus camera to fall on opposite slopes of the cornea. The resulting cornea-induced parallax creates a hyperstereoscopic effect that is evident when the sequential pair of photographs is viewed together. There are a variety of optical stereo viewers available for viewing side-by-side 35mm film images on a light box, or digital images on a computer monitor. All stereo viewing devices are designed to deliver the separate stereo images simultaneously but independently to each eye allowing the brain to fuse the pair.
Scanning Laser Ophthalmoscopy
Fluorescein angiography can also be recorded using a confocal scanning laser ophthalmoscope (cSLO) in place of the conventional fundus camera. This complex instrument uses a laser beam of the appropriate excitation wavelength to scan across the fundus in a raster pattern to illuminate successive elements of the retina, point-by-point.6,7
The laser can deliver a very narrow wavelength band for more efficient excitation of fluorescence than the flash illumination generated by a fundus camera flash tube. A confocal aperture is positioned in front of the image detector at a focal plane conjugate to the retina, effectively blocking non image-forming light. The confocal optical system and laser illumination combine to produce high contrast, finely detailed images. The laser scan rate is synchronized at a frame rate compatible with digital video display, providing a continuous high-speed representation of the flow dynamics of the retina and choroid. This can be especially useful when documentation of the very early filling stages is necessary, such as in identification of CNV feeder vessels. Both 30o and 50o auxiliary lenses are available for the cSLO; the wide-angle lens is primarily used for peripheral imaging in either diabetic retinopathy or venous occlusive disease. In addition, real-time montage software allows the operator to view on the screen a composite of multiple peripheral images as a single ultra-wide angle image. cSLO technology can also be used for indocyanine green (ICG) angiography of the choroidal vasculature, simultaneous fluorescein and ICG angiography, and for fundus autofluorescence imaging of retinal pigment abnormalities.
Proper sequencing of the angiographic series is essential in obtaining maximum diagnostic information. Color fundus photographs as well as black-and-white monochromatic green filter images are routinely taken as baseline views before administering the dye. The early transit phase is the most critical part of the angiogram and usually lasts less than a minute. Before injecting the dye, the illuminating beam of the fundus camera is centered within the dilated pupil. The angiographer then pre-focuses the camera on the appropriate area of interest. The dye is administered as a bolus injection, typically through a small gauge needle into an antecubital vein. The timer is started and photography commences. The arm-to-retina circulation time varies, but normally takes 10-12 seconds. Experienced angiographers anticipate the initial appearance of the dye and begin the photographic sequence before the dye is visible. Images are routinely captured at a rate of one frame per second until maximum fluorescence occurs. During this dynamic early phase only one eye can be captured. After completion of the early phase, photographs of the fellow eye or other areas of interest in the primary retina can be taken.
Over the next few minutes the appearance of the dye stabilizes and begins to slowly fade. The angiographer can capture appropriate views as necessary without the urgency needed during the early phase. Late phase photographs are taken as the dye dissipates, anywhere from 7 to 15 minutes after injection.
Many facilities develop disease specific protocols for both sequencing and field of view. For example, peripheral shots of the retina are routinely taken after the early transit in diabetic retinopathy, whereas the macula is the major area of interest in age-related macular degeneration and peripheral views are not usually necessary. The angiographer often adjusts the specific protocol based on visible changes that may occur as the angiogram progresses.
Quality Issues and the Role of the Angiographer
Some ophthalmologists perform their own angiography, but this is an exception rather than the rule. Most facilities employ a photographer or technician dedicated to performing ophthalmic photography procedures. Quality angiographic results rely on a number of factors. The skill of the angiographer and the optical and mechanical quality of the instrumentation can have a direct effect on results, but there are a number of common factors that can adversely affect angiographic quality. Media opacities can cause illumination artifacts and blurring of the images. Inadequate pupillary dilation reduces light reaching the retina, causing uneven illumination. Excess topical fluorescein staining of the cornea from the initial patient workup can compete with and degrade retinal fluorescence. Inadequate patient cooperation such as poor fixation or inability to hold steady during the procedure often results in loss of field definition during the important early transit phase. Extravasation of the dye not only causes discomfort to the patient, but the resulting incomplete dose reduces the amount of dye in the retinal vessels. Some of these causes are beyond the direct control of the angiographer, but every attempt should be made to minimize their detrimental effects in order for each angiogram to be of adequate and consistent diagnostic quality.
Since fluorescein angiography is a dynamic process, successful results depend on complete preparation before the dye is injected. Many angiographers follow a specific protocol or checklist to ensure that everything is ready. Good communication between the ophthalmologist and angiographer is essential to ensure that maximum diagnostic information is obtained. The photographic timing sequence and the angiographer’s ability to adapt to changing conditions are also important elements in producing quality angiographic results. Experience is invaluable, especially in managing the patient if complications occur during the critical early phase of the study.
Unfortunately, there is very little education available in fluorescein angiography. In the absence of formal education, certification plays an important role in developing competent practitioners in angiography. The Ophthalmic Photographers’ Society Inc. offers a voluntary certification program in fluorescein angiography that has established standards of competence in angiography. The Certified Retinal Angiographer (CRA) program was established in 1978. The program is accredited by the National Commission for Certifying Agencies and has certified over 800 individuals to date. Although certification is not mandatory, the CRA credential offers some assurance of competence and safety to both patient and physician.
The responsibility for injecting the dye sometimes falls to the angiographer or a technician. In some practice settings this makes sense. There are however, some legal issues associated with unlicensed personnel performing fluorescein injections.8 It is generally recommended that angiographic facilities check their current state or local laws regarding the credentialing requirements of personnel performing intravenous injections.9
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Fundamentals of Fluorescein
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- Kabachinski J. DICOM: key concepts—part II. Biomed Instrumen Technol. 39:292-294, 2005.
- Bidgood WD Jr, Horii SC. Introduction to the ACR-NEMA DICOM standard. Radiographics 12:345-355, 1992.
- Woon WH, Fitzke FW, Chester GH, et al. The scanning laser ophthalmoscope: basic principles and applications. J Ophthalmic Photography 12:17-23, 1990.
- Clark TM. Scanning laser ophthalmoscopes. In: Saine PJ, Tyler ME, eds. Ophthalmic Photography: Retinal Photography, Angiography and Electronic Imaging. 2nd ed. Boston, Butterworth-Heinemann, 2002:306-321.
- Ellis JH, Weber P. Legal issues arise when unlicensed personnel administer IV fluorescein. Argus Nov/Dec:28-31, 1995.
- Ophthalmic Photographers’ Society Standards of Practice. J Ophthalmic Photography; 21:26, 1999.