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Autofluorescence - Multicolor Imaging

Autofluorescence - Multicolor Imaging

May 25, 2022

Multicolor imaging (MCI) is a novel, noninvasive retinal imaging technique. Instead of white light used for conventional color fundus photography (CFP), MCI uses three laser lights to image the retinal layers.

MCI has shown promising imaging capabilities in various retinochoroidal diseases such as epiretinal membrane, age-related macular degeneration, and optic disc changes.

Fundus autofluorescence (FAF) demonstrates the functionality of the retinal pigment epithelium (RPE) while multicolor imaging(MCI), by means of its three incident wavelengths, provides insight into clinical changes at various levels of the retina and choroid in CSCR.

Advances in medical technology are bringing exciting changes to the field of ophthalmology. Through the innovation of industries and collaboration with doctors, these new technologies are helping to improve outcomes for laser eye surgery and to open up new ways of seeing for those with blindness.

The new technology in ophthalmology can improve outcomes and provide new treatment options for vision correction, IOP (intraocular pressure) reduction, and cataract surgery.

These innovations may change the options available to your local ophthalmologist when it comes to improving your vision and eye health.

New Advances in Medical Technologies for Ophthalmology

New ophthalmology exam tools, diagnostic imaging, and even vision replacement technology are changing the landscape for diagnosis and treatment of vision problems. Some examples of the recent technological advances in ophthalmology include:

Corneal Topography

By using non-invasive diagnostic imaging, corneal topography equipment maps the surface and shape of the cornea. 

By analyzing the curvature, thickness, and other details of the corneal surface, ophthalmologists can diagnose conditions such as keratoconus and can fit contact lenses to the unique characteristics of an individual.

Corneal topography is an essential diagnostic technology that creates a colored map of the cornea curvature with a specialized camera and digital analysis that highlights any abnormalities and guides treatment. Specific imaging methods include placido disc, Scheimpflug, and scanning-slit topography.

Optical Coherence Tomography (OCT)

By combining multiple diagnostic images through computerized analysis, Optical Coherence Tomography (OCT) creates images of the structure and blood flow of the retina. This non-invasive equipment eliminates the need for injected fluorescent dyes while creating very accurate and detailed images for review.

The detailed 3-D scans produced by an OCT machine can help ophthalmologists diagnose and treat diseases of the retina, including vessel occlusions, AMD, and diabetic retinopathy. These accurate images of the eye provide a clear picture of what is causing issues with vision, as well as tracking improvements or progression of retinal diseases.

Different Approaches to Visualizing Different Scenarios

Available modalities depend on white light from a standard flash nonconfocal camera, as in conventional fundus cameras (Canon and Topcon); a scanning laser with 2 wavelengths (Optos); slit-light confocal light-emitting diodes (LEDs; Eidon, CenterVue); broad line fundus images using blue, green, and red LEDs (Zeiss Clarus, Carl Zeiss Meditec); a scanning confocal laser with 3 wavelengths of blue, green, and near infrared (IR; Spectralis, Heidelberg Engineering); and a scanning confocal laser with 3 wavelengths of blue, green, and red (Scanning Laser Ophthalmoscope Mirante, Nidek).

The main difference between the normal fundus and multicolor images is the use of near IR reflectance instead of red and the confocal aperture. Once a normal fundus image is split into red-green-blue channels, the red image of the retinal pigment detachment shows high reflectance.

Meanwhile, the near IR (NIR) reflectance component of a multicolor image of the same case shows a dark area because of the absorption of the IR light. Thus, when the red channel is produced by IR light, only blue and green light are visualized.

The confocal aperture provides higher definition and sharp images. Moreover, as the focuses of the NIR light and the blue and green differ, the image colors can change depending on the degree of focus.

In 2 images obtained from the same patient on the same day with scan focuses of -2.37 D and -0.72 D, the details differed completely in sharpness and color.


Results from a number of studies have reported on the importance of multicolor images in identifying specific features such as pseudodrusen and atrophy compared with other imaging modalities.

The use of a confocal aperture is a key component in the superiority of multicolor images. The confocal aperture allows visualization of only the light from the area of interest and not that coming from the surrounding area.

This capability results in a markedly increased sharpness of the images. The sharpness obtained using the confocal aperture enables the identification of choroidal neovascularization, which is not visible using nonconfocal color fundus images or autofluorescence.

The use of single-wavelength green, blue, or NIR light coupled with the confocal aperture allows better definition of single structures such as the nerve fiber layer (blue) and retinal pigment epithelium (green), and deeper structures such as the choroidal vasculature.


This technology uses the fluorescence emanating from lipofuscin in the retinal pigment epithelial cells. Numerous available instruments can produce autofluorescence images; however, the blue light is obtained from the confocality of the instruments and green from other instruments.

Confocality is important in this case because all the autofluorescence light is collected from the entire eye, including the lens, which can interfere with fundus visualization. The use of confocality with the possibility to obtain only the light coming from the layer of interest permits visualization of the fundus autofluorescence.

For the green autofluorescence, the absorption and fluorescence from the lens are less, and instruments without a confocal aperture also can obtain an autofluorescence fundus image.                 The blue light visualizes the macular pigment, which is yellow and absorbs blue light.

The blue autofluorescence can be used to identify macular atrophy, pseudoholes, lamellar holes, and macular holes before and after surgery. For multiple evanescent white dot syndrome, an inflammatory disease, autofluorescence imaging shows bleaching of the characteristic white dots after treatment.

In inflammatory choroidal neovascularization, hyperautofluorescence is always apparent. In the expansion of serpiginous choroiditis, the enlarging area is always hyperautofluorescent before progressing to atrophy.

Multicolor imaging provides the sharpest images due to the confocal aperture and better visualization of some pathologies because of the IR reflectance component.

Autofluorescence imaging is the best technology to detect the retinal pigment epithelium and is important for the differential diagnosis in macular atrophy, macular holes, pseudoholes, and lamellar hole and inflammatory diseases.