82
retinal irradiance established by the American National
Standard Institute and other international standards
(22)
.
cSLO enables the acquisition of FAF images from wide
areas of the retina (55º with one frame and even larger
areas using the composite mode)
(20,22)
. Although lim-
ited by the optical properties of the human eye, SLO
succeeds in imaging the posterior pole with a high
contrast.
Currently there are three different cSLO sys-
tems for FAF imaging: the Heidelberg Retinal
Angiograph (HRA) (based on the HRA classic, HRA
2 and the Spectralis HRA) (Heidelberg Engineering,
Dossenheim, Germany); the Rodenstock cSLO
(RcSLO; Rodenstock, Weco, Düsseldorf, Germany);
and the Zeiss prototype SM 30 4024 (ZcSLO; Zeiss,
Obercochen, Germany). HRA is the only currently
commercially available system with the cSLO system
to capture FAF images. HRA uses an excitation wave-
length of 488 nm from an Argon laser or a solid-state
laser. A barrier filter with a short-wavelenght cutoff at
500 nm is inserted just opposite the detector, block-
ing the laser light and letting the autofluorescent
light through. Recently, it has been made possible to
acquire real time images, a technique known as real-
time averaging.
The Rodenstock cSLO and the Zeiss prototype SM
30 4024 have also been used to acquire clinical FAF
images. Both systems use an excitation wavelength of
488 nm (the same as the HRA), and barrier filters at
515 nm and 521 nm, respectively
(20,21,23)
.
Bellmann et al. have noticed marked contrast and
brightness differences as well as in the grey range (an
important marker of the image quality between the
different systems of cSLO). These limitations must be
taken into consideration when comparing images from
different cSLO systems
(24)
.
The default software of the HRA system normalizes the
pixel distribution of the final image in order to improve
the distribution of the FAF intensity. Even though this
final step facilitates the evaluation of the localized
topographic differences, it allows a relative estimation
of the intensities of the FAF. Thus, it should not be
used for quantitative calculation and absolute com-
parison between different FAF images. The normaliza-
tion of the average images can be easily turned off, and
brightness and contrast can be manually adjusted to
permit an adequate visualization of the distribution of
autofluorescence in areas with a very high or very low
signal in order to improve the visualization of small
details.
The detection of FAF is limited by its low intensity
(approximately two orders of magnitude lower than the
peak background fluorescence of an ordinary
fluores-
cein angiography), and by the autofluorescence charac-
teristics of the anatomic structures of the eye, including
those of the optical media, especially of the lens
(16)
.
2. Imaging methods
2.1 Fundus spectrophotometry
Fundus spectrophotometry was developed by Delori
et al. and was designed to determine the spectrum of
excitation and fluorescence emission from small areas
of the retina (2º diameter)
(17,18)
. The authors were able
to determine the amount of autofluorescence and com-
pare it with in vitro fluorescence microscopy. They
found out that the spectrum of in vivo excitation was
slightly broader, and peaked at a longer wavelength
than those of A2E and native LF. The authors con-
cluded that considering the spatial distribution, spec-
tral characteristics and age relationship, LF is the main
source of fluorescence in the FAF in vivo
(18)
.
Presently, two systems are available to examine the
autofluorescence of the human eye in vivo in the clini-
cal practice: confocal scanning laser ophthalmoscope
and fundus camera.
2.2 Scanning laser ophthalmoscope
Confocal scanning laser ophthalmoscope (cSLO) was
originally developed by Webb et al. using a low-energy
laser source to scan the retina in two directions: termed
as x and y
(19)
. The confocal nature of the optics ensures
that the reflectance and fluorescence correspond to the
same focal plane. cSLO overcomes the limitations of
the low-intensity signal of FAF and the lens interfer-
ences. The defocused light is almost completely sup-
pressed, thus reducing the autofluorescence from the
optical media anterior to the retina, such as the lens or
the cornea.
In order to reduce the background noise and to increase
the contrast of the image, a series of FAF images are
usually recorded
(20,21)
. Following the aligning of the
images in order to correct the movement of the eye
during the acquisition, the final image is calculated
(usually from 4-32 frames) and the values of the pixels
are normalised. The FAF image can be obtained with
low excitation energies within the limits of maximum
