CLINICAL SCIENCES

Simultaneous Indocyanine Green

and Fluorescein Angiography Using
a Confocal Scanning Laser Ophthalmoscope
William R. Freeman, MD; Dirk-Uwe Bartsch, PhD; Arthur J. Mueller, MD, PhD;
Alay S. Banker, MD; Robert N. Weinreb, MD

Background: Fluorescein and indocyanine green (ICG)
angiography are both useful in the diagnosis and treat-
ment of many retinal diseases. In some cases, both tests
must be performed for diagnosis and treatment; how-
ever, performing both is time-consuming and may re-
quire multiple injections.
Methods: We designed a compact digital confocal scan-
ning laser ophthalmoscope to perform true simulta-
neous fluorescein and ICG angiography. We report our
experience using the instrument to perform 169 angio-
grams in 117 patients.
Results: There were no unexpected adverse effects from
mixing the dyes and administering them in 1 injection.
An entire examination, including fundus photography,
fluorescein angiography, and ICG angiography, could be
performed in 45 minutes. It was possible to study dif-
ferences in fluorescein patterns by comparing identi-
cally timed frames and to find cases in which ICG or fluo-
rescein was optimal in visualizing retinal and subretinal
structures. Confocal optical sections in the depth (z) di-
mension allowed viewing in different planes. It was pos-
sible to overlay ICG and fluorescein images or compare
them side-by-side using a linked cursor. Digital trans-
mission of the images was also performed.
Conclusions: Simultaneous ICG and fluorescein angi-
ography can be performed rapidly, safely, and conve-
niently. The availability of simultaneous angiography will
allow critical determination of the relative advantages and
disadvantages of both types of angiography.
Arch Ophthalmol. 1998;116:455-463

From the Department of
Ophthalmology, Shiley Eye
Center, University of
California, San Diego.
F
LUORESCEIN angiography is a
useful tool for the diagnosis
of many retinal diseases. It
provides diagnostic as well as
treatment information by al-
lowing visualization of the retinal and cho-
roidal vasculature. Angiography allows
identification of leakage of the small fluo-
rescein molecule in pathological states.
More recently, indocyanine green (ICG)
angiography has been developed. This
molecule is larger (molecular weight, 775
d vs 332 d for fluorescein) and more pro-
tein-bound in plasma than is fluorescein
and fluoresces in the infrared spectrum.
Initially, ICG angiography was per-
formed with infrared photographic film.
However, the poor sensitivity of film
coupled with the relatively weak fluores-
cence properties of the dye caused this
method to be abandoned.1-3 The strong
binding of ICG dye to plasma proteins re-
sults in slow leakage as compared with
fluorescein and reduces the amount of ex-
travascular fluorescence available for im-
aging. Digital video cameras have been
used to capture images for ICG angiogra-
phy. This has made ICG angiography a
useful clinical diagnostic tool, particu-
larly for imaging of subretinal neovascu-
lar membranes in cases where such mem-
branes can not be adequately imaged with
fluorescein angiography.4-8
For editorial comment
see page 521
Another approach to fluorescence an-
giography (using either fluorescein or ICG)
is the scanning laser ophthalmoscope.9 This
instrument has come into common clini-
cal practice because of its commercializa-
tion in recent years.10 Advantages of the
scanning laser ophthalmoscope include the
ability to use an excitation light that scans
the retina, allowing more intense excita-
tion (thus providing a stronger emission sig-
nal) while still using safe levels of illumi-
nation. This is possible because the
scanning beam illuminates each area point
of the retina for only 0.1 to 0.7 microsec-
onds. Scanning laser ophthalmoscope an-
giography gives temporal information that

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 MATERIALS AND METHODS
We used a confocal scanning laser ophthalmoscope with
2 laser light sources to illuminate the retina (Heidelberg
Retina Angiograph, Heidelberg Engineering Inc, Carls-
bad, Calif). The instrument uses 2 lasers with 3 wave-
lengths as light sources for scanning fundus illumination.
An argon-ion laser (488 nm and 514 nm wavelength) was
used to provide red-free photographs (green, 514 nm) and
blue light was used for excitation during fluorescein an-
giograms (blue, 488 nm). The second laser was a diode la-
ser (795 nm wavelength) that provided illumination to ex-
cite the ICG dye, which fluoresces at 835 nm. The
instrument was operated in a tight confocal imaging mode
and acquired up to 12 simultaneous frame pairs per sec-
ond. For each video line of the angiogram, the forward scan
was used for one angiogram and the backward scan was
used for the other angiogram. Thus, the time separation
between corresponding lines of the 2 angiograms was on
the order of 0.1 milliseconds; each frame pair was ac-
quired in 0.08 seconds. The instrument allowed acquisi-
tion of 12 frame pairs per second with 256 3 256 pixel and
8 bit per pixel intensity quantitation.
The images were recorded in the confocal mode12,16,17,18
while the photographer moved the plane of focus in the
depth (z) dimension during the angiogram to optimally im-
age the structures of interest. The chromatic aberration of
the human eye caused a 1-diopter (D) focus shift between
the blue-green fluorescein angiogram image plane and the
infrared ICG angiogram image plane. The photographer se-
lected a focal plane that allowed in-focus imaging of the
retinal vasculature in the fluorescein angiogram, which
placed the ICG image plane 300 µm deeper in the retina
and choroid.
is richer than still (“digital”) imaging systems because true
video allows imaging at rates of 20 to 30 frames per sec-
ond. This allows more detail to be seen in the transit phases
and may give better information about patterns of leak-
ing vessels. Furthermore, the illuminating beam is mono-
chromatic and the intensity of fluorescence may be higher
because of the scanning laser ophthalmoscope beam and
the fact that point-by-point illumination is used.10 Stereo-
scopic imaging is also possible using scanning laser oph-
thalmoscope techniques.11
We recently described the use of a confocal scan-
ning laser ophthalmoscope to perform tomographic im-
aging of macular diseases.12 By optically isolating the im-
age plane to a narrow plane in the depth (z) dimension,
depth information and measurements could be ob-
tained. Subsequently, we adapted the scanning laser to-
mograph to perform ICG angiography.13,14 The instru-
ment allowed better discrimination against out-of-focus
objects (tomography) and provided higher contrast than
other systems. The simple optical path and optimized ex-
citation and emission detection systems improved the sen-
sitivity of the instrument so that 70% of the photons ex-
iting the eye could be detected. This allowed excellent
visualization of vessels in the late stages (45 minutes) of
the ICG angiogram without the use of a second (“land-
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©1998 American Medical Association. All rights reserved.
PATIENT EXAMINATIONS
We performed 169 angiograms in 117 patients with age-
related macular degeneration, ocular melanoma, and other
retinal diseases. The patients were studied after a complete
ophthalmic examination that included fundus photogra-
phy, slitlamp examination, and indirect ophthalmoscopy. We
injected 2 mL of a mixture of 25 mg of ICG and 500 mg of
sodium fluorescein intravenously. This was prepared using
sterile commercially available dyes suitable for intravenous
injection. The liquid sodium fluorescein (25% solution in 2
mL; Fluorescite, Alcon, Fort Worth, Tex) was placed into a
vial containing sterile ICG powder (CardioGreen, Becton
Dickinson, Cockeyville, Md) and the ICG was dissolved in
the liquid fluorescein. No precipitates were seen. This re-
sulted in a sterile solution containing 25 mg of ICG and 500
mg of sodium fluorescein. The patients underwent imaging
in the early phase (0-2 minutes after injection), the mid phase
(3-5 minutes for sodium fluorescein, 3-15 minutes for ICG),
and late phase (10-12 minutes for sodium fluorescein, 40-45
minutes for ICG). All patients were informed as to the risk
and benefit factors of all procedures. The images were ana-
lyzed by 2 ophthalmologists (W.R.F. and A.J.M.).
IMAGE STORAGE AND RETRIEVAL
The images were stored digitally in the RAM of the com-
puter during acquisition and subsequently transferred onto
the hard disk. The still frames of a typical angiogram se-
quence (n=50 frames) required 3.2 megabytes of hard disk
memory. Video sequences (20 frames per second) re-
quired 13 megabytes for each 10-second sequence of video.
Using 40 megabytes of RAM, we allowed 30 seconds of live
video storage before moving the data to permanent hard
disk storage.
mark”) injection of ICG dye.5 This instrument was the
first completely digital instrument to allow scanning la-
ser video angiography. Image capture and processing is
done digitally so that no information is lost at any stage
of processing, image manipulation, or transmission.
Although ICG angiography may be advantageous in
certain cases of subretinal neovascularization and in di-
agnosing other disorders,4,5,7 one of its drawbacks is the
long period of time required for angiography (45 min-
utes) and the need to obtain a second angiogram after a
fluorescein angiogram is obtained. The process of ob-
taining a fluorescein and ICG angiogram sequentially is
time consuming and requires 2 or 3 (if a landmark in-
jection is used) injections for each patient for comple-
tion of the set of studies. Moreover, the angiograms are
performed at different times, making it difficult to know
if differences in fluorescence leakage patterns are due to
differences in properties of the dyes or in the quality of
the photographs. This may be one reason why different
investigators vary in their assessment of the additional
incremental benefit of ICG over fluorescein angiogra-
phy.4,10,14-16 To determine if high-quality simultaneous im-
ages from fluorescein and ICG angiograms could be ob-
tained, we modified a small, digital, personal computer–
based confocal scanning laser ophthalmoscope to allow
 A B

C D

Figure 1. Confocal fluorescein–indocyanine green (ICG) angiogram showing hyperfluoresence and early leakage due to subretinal neovascular membrane at 56.03
seconds. The fluorescein angiogram shows early leakage (A) but the vascular membrane is not well defined. The ICG frame (B) shows a vascular plexus (top of
cursor). The cursors in A and B frames are at the same location on the fundus. Late frames on the fluorescein (C) and ICG (D) angiograms show leakage at 42
minutes 15 seconds. Note that the dark outlines of retinal vessels are clearly seen in the late ICG frame, allowing localization of retinal vascular landmarks without
a second (“landmark”) injection.

simultaneous imaging after a single intravenous injec-
tion of both ICG and fluorescein. We do not advocate
performing simultaneous angiography on every patient;
rather, our goal was to determine whether such angiog-
raphy could be performed and to evaluate the character-
istics of confocal simultaneous angiography.
RESULTS
CLINICAL ADVANTAGES
One hundred sixty-nine angiograms were performed with
no serious adverse reactions. Preparation of the dye mix-
ture from the 2 commercially available sterile prepara-
tions was performed without problems. A saline solu-
tion “flush” injection was not necessary to obtain good
image quality. The time necessary for an entire study was
45 minutes and only 1 injection per patient was needed.
Red-free color images were also obtained using the green
wavelength of the argon laser. Infared fundus photo-
graphs were obtained using the diode laser. It was not
necessary to reinject ICG dye to image retinal vessels in
the late phase of the study. The retinal vessels were vis-
ible at the 40-minute time point owing to the sensitivity
of the detector, which allowed visualization of the back-
ground choroidal ICG dye fluorescence. This allowed im-
aging of the retinal vessels as dark linear structures sil-
houetted by this background fluorescence13 (Figure 1).

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 A B

Figure 2. Confocal fluorescein–indocyanine green (ICG) angiogram in an eye with a choroidal nevus, showing venous laminar phase at 11.42 seconds along the
inferotemporal branch retinal vein. The location of the beginning of the laminar flow is the same in both fluorescein (A) and ICG (B) angiograms and occurs at the
junction of the third small tributary vein from the optic nerve head in the photographs.

TIME COURSE OF APPEARANCE OF BOTH DYES
Analysis of patients in whom rapid sequences of early
frames were performed showed that the transit of ICG
and fluorescein to the eye appeared to be simultaneous.
This was best seen in the early laminar flow or arterial
filling phases where arterial filling or localized venous
laminar flow patterns could be seen. In these cases, the
patterns of vascular filling over time appeared to be the
same with both dyes (Figure 2). We found that it was
possible to artificially make either dye appear to fluo-
resce earlier by changing the gain settings on the instru-
ment. We did not perform absolute calibration of the emit-
ted light from each point on the fundus. We found that
it was easy to study the differences in fluorescence pat-
terns, lesion localization, and leakage patterns using ICG
and fluorescein with the simultaneously acquired im-
ages (Figure 2).
CLINICAL UTILITY IN SUBRETINAL
NEOVASCULARIZATION AND OTHER DISEASES
The production of true simultaneous images negated the
possibility that one of the angiograms was of higher qual-
ity than the other and that this allowed better visualiza-
tion of pathological states. The potential reasons that one
study might be of better quality include better centering
of the camera image pathway in the pupil, less patient
movement, better focus, more correct exposure, less blink-
ing, and other factors. Using the simultaneous angio-
gram, in some cases ICG showed a subretinal neovascu-
lar membrane better than fluorescein and in other cases
fluorescein showed it better than ICG (Figure 3). Only
a larger study can determine the relative sensitivity and
specificity of the 2 techniques. Our review of the images
suggests that both studies are needed in difficult cases.
It is interesting that in some cases, feeder vessels can be
seen in ICG and not in fluorescein angiogram frames
(Figure 4).
We found that the ability to view multiple images
or video-rate images provided more information than
single frames. A pseudostereo image was possible by slowly
moving the instrument in the horizontal meridian.11 The
information density included horizontal resolution and
a temporal component. During the study, frames were
selectively focused at different planes because of the con-
focal nature of the detection system. The ability to pro-
cure images at rapid rates allowed us to select the most
optimal frame for determining and guiding treatment.
CONFOCALITY
The use of confocal optics had 2 effects. It allowed im-
aging in a relatively narrow plane of focus and it also in-
creased the contrast considerably. This improved the qual-
ity of the images.13 In addition, the ability to move the
plane of focus in depth allowed the physician to discern
the optimal plane of focus for visualizing the pathologi-
cal structures of interest. As previously described,13 the
higher contrast of this system allowed visualization of the
retinal vasculature superimposed on the background ICG
fluorescence in 40-minute late frames of the ICG angio-
grams (Figure 1). In addition, we were able to show the
depth location of subretinal neovascular membranes by
scanning at different planes of focus (Figure 5).
REGISTRATION AND COMPARISON
OF SIMULTANEOUS IMAGES
We studied 2 methods for comparing the simultaneous
ICG and fluorescein images. Color-coding the images
(fluorescein in green and ICG in red) allowed overlay of

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 A B

C D

Figure 3. Confocal fluorescein–indocyanine green (ICG) angiogram showing an organizing pigment epithelial detachment with underlying subretinal neovascular
membrane seen better on fluorescein then ICG images. Hyperfluoresence and early leakage of dye are seen more impressively at 1 minute 5 seconds on the
fluorescein (A) than ICG (B) frames. Later frames of the fluorescein (C) and ICG (D) studies also show more leakage and dye pooling in the fluorescein study (6
minutes 47 seconds).

the 2 images (Figure 6). We also were able to study the
2 monochromatic gray-scaled images side by side, elec-
tronically linked with a cursor. The cursor location in
the 2 images always corresponded to the identical loca-
tion in the fundus. This allowed simultaneous viewing
of a given point on the fundus in both ICG and fluores-
cein images (Figure 7). We found this less confusing
and more precise than the dual-color method. This
method was possible only because each line of the an-
giogram was scanned both for ICG and fluorescein im-
ages in a quasi-simultaneous fashion and correspond-
ing points could be determined. There was no image
manipulation involved in performing this electronic link-
ing between the 2 images.
IMAGE STORAGE, TRANSFER, AND RETRIEVAL
The output of the instrument was digital. Thus each im-
age entailed 256 3 256 bytes plus 8-bit gray-scale infor-
mation at each location. Each location of the fundus was
imaged twice so that each image pair (ICG and fluores-
cein) was approximately 130 kilobytes. The completely
digital nature of the output allowed storage of all of the
acquired information electronically without any loss of
detail. The images could be stored using the software de-
signed for the instrument or could be loaded into any com-
mercially available software on any platform, either PC
or Macintosh. Several selected pairs of images could be
easily transported on a single floppy disk to a second com-

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©1998 American Medical Association. All rights reserved.

 A B C

Figure 4. A, Confocal fluorescein–indocyanine green (ICG) angiogram showing hyperfluoresence and early fluorescein leakage in the foveal area at 53.04 seconds.
B, Simultaneous ICG frame shows a feeder vessel perfusing a round subretinal neovascular membrane. C, 32 magnification of (B) shows details of feeder vessel
and subretinal neovascular membrane. In all 3 images the cursors are in precisely the same location because the images are digitally aligned pixel by pixel. The
cursors are linked and correspond to the same location for each simultaneous pair.

A B

C D

Figure 5. Frames from an indocyanine green (ICG) image series showing a subretinal neovascular membrane. The confocal series was taken at varying depths. A,
−6.0 diopters (D); B, −4.0 D; C, −2.5 D; and D, +2.0 D. The subretinal neovascular membrane is seen at the −2.25 D plane as are other choroidal details.

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 A B

Figure 6. A, Simultaneous fluorescein and indocyanine green (ICG) angiogram frames of a patient with angioid streaks displayed side by side. B, Color-coded
information from both frames in 1 image. Green indicates fluorescein fluorescence and red indicates ICG.

puter in a laser treatment room. We also were able to trans-
port images via Internet connections (approximately 5
seconds per image pair) to remote sites anywhere in the
world. Video information was stored on portable hard
disk drives and a variety of other media at low cost. At
our institution, we chose to use Ethernet cable to trans-
port image pairs at times of approximately 1 second per
ICG/fluorescein pair from the angiography suite to the
laser suite.
Red-free photographs were also obtained with the
green 514.5-nm channel of the argon laser and infrared pho-
tographs were procured with the diode laser wavelength.
These showed subretinal structures such as drusen more
clearly then color or red-free fundus photographs.19
These photographic images were not simultaneous
but were taken at the same magnification and were suit-
able for performing overlays on angiographic images, as
are used in posttreatment assessment in eyes treated for
subretinal neovascularization.9,20 Infrared fundus pho-
tographs could also be performed with the instrument.
COMMENT
Simultaneous confocal ICG and fluorescein angiogra-
phy using a confocal scanning laser ophthalmoscope has
several advantages. Clinically, we noted no serious ad-
verse reactions in a series of 169 simultaneous ICG/
fluorescein injections. The time required to perform the
entire study was considerably shorter than first perform-
ing a fluorescein study, reviewing it, and subsequently
performing the ICG study. In addition, the confocal na-
ture of the instrument allowed for extremely high con-
trast in the later images, which allowed visualization of
retinal vessels at the late time points and obviated the need
for a landmark injection of fluorescein. Thus, simulta-
neous angiography is beneficial both from the patient’s
perspective as well as that of the ophthalmologist’s of-
fice staff; only 1 injection is needed and in 40 minutes
fundus photography, ICG angiography, and fluorescein
angiography can be completed and reviewed to diag-
nose and guide treatment. In general, we performed si-
multaneous angiography in cases in which prior fluo-
rescein angiography did not or was not expected to reveal
sufficient information for treatment or diagnosis. A sa-
line solution flush injection was not used as high-
quality images were obtained without it.
The ability to perform simultaneous angiography was
demonstrated by Bischoff et al,21 who described a pro-
totype simultaneous ICG and fluorescein angiogram based
on a scanning laser ophthalmoscope. Their instrument
differed from the instrument we describe. Their instru-
ment acquired true simultaneous images of the fundus
with 2 continuous laser light sources and 2 light detec-
tors. It was a prototype and required the presence of a
complicated computer setup, 2 video recorders, and 2
monitors, filling half a room. The set-up of the instru-
ment in our study switches laser light sources during the
acquisition of a single video line to capture quasi-
simultaneous images (time separation, 0.1 millisec-
onds). The light exposure is limited as sources are never
simultaneously active during the scan. In addition, the
present instrument allows acquisition of red-free and in-
frared fundus photographs, as well as storing all images
digitally so that any image can be recalled or trans-
ported at any time or location without any loss of image
quality. The use of a PC computer platform allows the
use of any software system and great flexibility in image
storage and processing. It is important to realize that in-
formation density not only includes horizontal resolu-
tion but also a time component. By shifting planes of fo-
cus and angle of imaging, one can often see better details
of the structure of interest and can also choose the indi-
vidual frame that best shows the pathological feature of
interest or the one that must be projected to guide laser
therapy. Other advantages of video imaging include the
ability to see moving particles in the fluorescein images;
this may enhance the visualization of subretinal neovas-
cular membranes.22
There has been some controversy regarding the is-
sue of transit times of the 2 fluorescent dyes.21,22 We found

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 A B C

D E F

G H

Figure 7. Confocal fluorescein–indocyanine green (ICG) angiogram of a patient with pseudoxanthoma elasticum and angioid streaks. The cursor marks identical
locations on the simultaneous fluorescein (A, C, E, and G) and ICG (B, D, F, and H) frames. All images are in the early phase of the angiogram at 2 minutes 14.82
seconds. A and B, The junction of a retinal artery and the disc margin, beneath which there is hyperfluorescence due to an angioid streak. This is not yet seen on
the ICG frame. C and D, A retinal artery, which is in an identical location on both the fluorescein and ICG frames as marked by the linked cursor. E and F, The
cursor moved again, this time to a circumferential area of hyperfluoresence seen on both the fluorescein and ICG frames corresponding to an angioid streak. In
operation, moving the cursor on one frame moves it on the other, allowing precise identification of corresponding areas on simultaneous ICG and fluorescein
frames. G and H, The late fluorescein images6 and ICG14; the angioid streaks are much more visible on the ICG image, as is the reticulated appearance of Bruch
membrane.

both dyes arrived simultaneously in the early phase of
our study. There is no reason for the dyes to arrive at dif-
ferent time points.23,24 When the relative gains of our de-
tectors were equal, there was no evidence that one of the
dyes transited faster than the other.
We found that it was easy to study the differences
in fluorescence patterns and lesion localization using ICG
and fluorescein with the simultaneously acquired im-
ages. We preferred the linked cursor software mode to
critically and simultaneously compare locations of the

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ICG and fluorescein images. Color-coding of the 2 im-
ages with display of both images simultaneously could
also be performed. We recognize that treatment based
on ICG fluorescence has not been studied in random-
ized clinical trials. Clearly this is necessary, but the abil-
ity to obtain both studies simultaneously should be ad-
vantageous in designing such a trial. Using simultaneous
angiography is the only way to determine the relative value
of each type of angiography. Only through a large study
of patients with a variety of retinal diseases using simul-
taneous ICG and fluorescein angiography will it be pos-
sible to critically determine the differences in vascular
imaging and leakage patterns and their clinical signifi-
cance.25 We do not advocate the use of simultaneous an-
giography for all patients. However, in those patients in
whom conventional fluorescein angiography does not al-
low a conclusive analysis, simultaneous angiography
seems to offer additional insight.
Accepted for publication November 26, 1997.
Supported by a grant from the Stern Foundation, La
Jolla, Calif (Dr Freeman), the Whitaker Foundation, Ross-
lyn, Va (Dr Bartsch), and Deutsche Forschungsgemein-
schaft, Bonn, Germany (Dr Mueller).
Reprints: William R. Freeman, MD, UCSD Shiley Eye
Center, 9415 Campus Point Dr, La Jolla, CA 92093-0946.
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