In vivo imaging of retinal pigment epithelium cells in age related macular degeneration

Abstract

Morgan and colleagues demonstrated that the RPE cell mosaic can be resolved in the living human eye non-invasively by imaging the short-wavelength autofluorescence using an adaptive optics (AO) ophthalmoscope. This method, based on the assumption that all subjects have the same longitudinal chromatic aberration (LCA) correction, has proved difficult to use in diseased eyes, and in particular those affected by age-related macular degeneration (AMD). In this work, we improve Morgan's method by accounting for chromatic aberration variations by optimizing the confocal aperture axial and transverse placement through an automated iterative maximization of image intensity. The increase in image intensity after algorithmic aperture placement varied depending upon patient and aperture position prior to optimization but increases as large as a factor of 10 were observed. When using a confocal aperture of 3.4 Airy disks in diameter, images were obtained using retinal radiant exposures of less than 2.44 J/cm 2 , which is ~22 times below the current ANSI maximum permissible exposure. RPE cell morphologies that were strikingly similar to those seen in postmortem histological studies were observed in AMD eyes, even in areas where the pattern of fluorescence appeared normal in commercial fundus autofluorescence (FAF) images. This new method can be used to study RPE morphology in AMD and other diseases, providing a powerful tool for understanding disease pathogenesis and progression, and offering a new means to assess the efficacy of treatments designed to restore RPE health. L. Dunaief, J. Z. Baffi, and J. Ambati, "DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration," Nature 471(7338), 325-330 (2011).

Automated segmentation and quantitative study of retinal pigment epithelium cells for photoacoustic microscopy imaging

“…6(c), a statistical analysis of the cell area is presented. The area distribution is centered at 200 μm 2 , which is similar to the result of the normal human eye [23] . The relationship between the cell area and intensity is shown in Fig.…”

“…To acquire the RPE cell features such as morphology and component content, accurate cell detection and segmentation in cellular images are important. In previous work, several semi-automatic and automatic cell segmentation methods have been used for cell evaluation, and they acquired remarkable results in the images of corneal endothelium, photoreceptor, and RPE cells [18][19][20][21][22][23][24] , using two-photon fluorescence microscopy, confocal microscopy, and adaptive optics retinal imaging. As these imaging results cannot provide melanin content information of RPE cells, an automatic RPE cell segmentation algorithm for PAM imaging is necessary.…”

Imaging individual neurons in the retinal ganglion cell layer of the living eye

“…Experiments were carried out on three different AOSLO systems capable of simultaneous confocal and off-axis detection: a human research AOSLO (48,49), a compact commercial prototype human AOSLO (50), and a nonhuman primate two-photon AOSLO (TPAOSLO) (26,51). Some humans were imaged on both systems, permitting comparison between systems, but some major differences between them prevented direct comparisons (see SI Materials and Methods for details).…”

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

“…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.…”