In Vivo Imaging of the Human Retinal Pigment Epithelial Mosaic Using Adaptive Optics Enhanced Indocyanine Green Ophthalmoscopy

Abstract

PurposeThe purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye.MethodsA customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out.ResultsThe AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection.ConclusionsAdaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells.

Noninvasive near infrared autofluorescence imaging of retinal pigment epithelial cells in the human retina using adaptive optics

“…Taking this into consideration, our AO-IRAFbased measurements are comparable to existing ex vivo measurements of RPE cell density and spacing from histological studies [33][34][35][36][37][38][39][40][41][42] as well as to in vivo measurements using other imaging modalities [13][14][15]22]. At the fovea, our density results are lower than Gao and Ach's measurements, while higher than the results of Watzke and Panda-Jonas (Fig.…”

“…2). RPE cells visualized using darkfield have also been previously shown to colocalize with two other modalities (AO short-wavelength autofluorescence [12] and AO-ICG [15]). This is consistent with a recent report that AO-IRAF directly colocalizes with AO short-wavelength autofluorescence [17].…”

“…A custom-built adaptive optics scanning light ophthalmoscope [18] that had been previouslymodified to detect fluorescence light from indocyanine green (ICG) (described in more detail in [15]) was further modified to improve detection of IRAF light, which shares similar excitation and emission bands. The excitation and emission spectra of our instrument were designed to closely match those of a commercially-available instrument with IRAF imaging capabilities (see next paragraph).…”

“…Power levels were fixed for each imaging session, but varied from session to session due to instrument alignment. Since the AO-IRAF signal is at least several-fold weaker than the AO-ICG signal [15], detection of fluorescent light in the 810-830 nm band was further enhanced by redesigning our computer-controlled fixation system [20] to utilize a large dichroic (cut-off wavelength 596 nm, product FF596-Di01, Semrock, Rochester, NY), which transmits a higher percentage of light in the 810-830 nm band than the 90/10 pellicle beamsplitter that we previously utilized for our AO fixation system. We also increased the size of the confocal pinhole that was used for detection of IRAF light from 3 Airy Units to either 6 or 8 Airy Units.…”

“…When combined with imaging platforms, AO ophthalmoscopes have the capabilities to resolve fine cellular details of the living human retina [9,10]. There are now a number of approaches that utilize AO for imaging RPE cells, based on capturing intrinsic scattering of light [11,12], generating contrast derived from organelle motility [13], visualizing autofluorescence of lipofuscin [14], and imaging fluorescent labeling by intravenously-injected indocyanine green dye [15]. Recently, we and others have independently reported that the integration of AO with IRAF can also be used to resolve individual RPE cells [16,17].…”