Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy

In vivo two-photon imaging through the pupil of the primate eye has the potential to become a useful tool for functional imaging of the retina. Two-photon excited fluorescence images of the macaque cone mosaic were obtained using a fluorescence adaptive optics scanning laser ophthalmoscope, overcoming the challenges of a low numerical aperture, imperfect optics of the eye, high required light levels, and eye motion. Although the specific fluorophores are as yet unknown, strong in vivo intrinsic fluorescence allowed images of the cone mosaic. Imaging intact ex vivo retina revealed that the strongest two-photon excited fluorescence signal comes from the cone inner segments. The fluorescence response increased following light stimulation, which could provide a functional measure of the effects of light on photoreceptors.     

In vivo two-photon imaging of the mouse retina

Though in vivo two-photon imaging has been demonstrated in non-human primates, improvements in the signal-to-noise ratio (SNR) would greatly improve its scientific utility. In this study, extrinsic fluorophores, expressed in otherwise transparent retinal ganglion cells, were imaged in the living mouse eye using a two-photon fluorescence adaptive optics scanning laser ophthalmoscope. We recorded two orders of magnitude greater signal levels from extrinsically labeled cells relative to previous work done in two-photon autofluorescence imaging of primates. Features as small as single dendrites in various layers of the retina could be resolved and predictions are made about the feasibility of measuring functional response from cells. In the future, two-photon imaging in the intact eye may allow us to monitor the function of retinal cell classes with infrared light that minimally excites the visual response.OCIS codes: (330.4460) Ophthalmic optics and devices, (180.4315) Nonlinear microscopy, (170.0110) Imaging systems     

Adaptive optics fluorescence lifetime imaging ophthalmoscopy of in vivo human retinal pigment epithelium

The intrinsic fluorescence properties of lipofuscin – naturally occurring granules that accumulate in the retinal pigment epithelium – are a potential biomarker for the health of the eye. A new modality is described here which combines adaptive optics technology with fluorescence lifetime detection, allowing for the investigation of functional and compositional differences within the eye and between subjects. This new adaptive optics fluorescence lifetime imaging ophthalmoscope was demonstrated in 6 subjects. Repeated measurements between visits had a minimum intraclass correlation coefficient of 0.59 Although the light levels were well below maximum permissible exposures, the safety of the imaging paradigm was tested using clinical measures; no concerns were raised. This new technology allows for in vivo adaptive optics fluorescence lifetime imaging of the human RPE mosaic.     

Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina

Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.OCIS codes: (170.4460) Ophthalmic optics and devices, (110.4500) Optical coherence tomography, (110.1080) Active or adaptive optics, (170.0110) Imaging systems, (330.7324) Visual optics, comparative animal models, (170.4470) Ophthalmology     

Imaging translucent cell bodies in the living mouse retina without contrast agents

The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse retina.OCIS codes: (170.4460) Ophthalmic optics and devices, (330.4300) Vision system - noninvasive assessment, (110.1080) Active or adaptive optics, (330.7324) Visual optics, comparative animal models     

Measurement and correction of transverse chromatic offsets for multi-wavelength retinal microscopy in the living eye

A special challenge arises when pursuing multi-wavelength imaging of retinal tissue in vivo, because the eye’s optics must be used as the main focusing elements, and they introduce significant chromatic dispersion. Here we present an image-based method to measure and correct for the eye’s transverse chromatic aberrations rapidly, non-invasively, and with high precision. We validate the technique against hyperacute psychophysical performance and the standard chromatic human eye model. In vivo correction of chromatic dispersion will enable confocal multi-wavelength images of the living retina to be aligned, and allow targeted chromatic stimulation of the photoreceptor mosaic to be performed accurately with sub-cellular resolution.OCIS codes: (110.1080) Active or adaptive optics, (130.2035) Dispersion compensation devices, (170.5810) Scanning microscopy, (330.5510) Psychophysics, (330.7327) Visual optics, ophthalmic instrumentation     

Adaptive optics SLO/OCT for 3D imaging of human photoreceptors in vivo

We present a new instrument that is capable of imaging human photoreceptors in three dimensions. To achieve high lateral resolution, the system incorporates an adaptive optics system. The high axial resolution is achieved through the implementation of optical coherence tomography (OCT). The instrument records simultaneously both, scanning laser ophthalmoscope (SLO) and OCT en-face images, with a pixel to pixel correspondence. The information provided by the SLO is used to correct for transverse eye motion in post-processing. In order to correct for axial eye motion, the instrument is equipped with a high speed axial eye tracker. In vivo images of foveal cones as well as images recorded at an eccentricity from the fovea showing cones and rods are presented.OCIS codes: (170.3890) Medical optics instrumentation, (110.1080) Active or adaptive optics, (170.4470) Ophthalmology, (330.5310) Vision - photoreceptors, (110.4500) Optical coherence tomography     

Adaptive optics with pupil tracking for high resolution retinal imaging

Adaptive optics, when integrated into retinal imaging systems, compensates for rapidly changing ocular aberrations in real time and results in improved high resolution images that reveal the photoreceptor mosaic. Imaging the retina at high resolution has numerous potential medical applications, and yet for the development of commercial products that can be used in the clinic, the complexity and high cost of the present research systems have to be addressed. We present a new method to control the deformable mirror in real time based on pupil tracking measurements which uses the default camera for the alignment of the eye in the retinal imaging system and requires no extra cost or hardware. We also present the first experiments done with a compact adaptive optics flood illumination fundus camera where it was possible to compensate for the higher order aberrations of a moving model eye and in vivo in real time based on pupil tracking measurements, without the real time contribution of a wavefront sensor. As an outcome of this research, we showed that pupil tracking can be effectively used as a low cost and practical adaptive optics tool for high resolution retinal imaging because eye movements constitute an important part of the ocular wavefront dynamics.OCIS codes: (110.1080) Active or adaptive optics, (100.4999) Pattern recognition, target tracking, (170.4460) Ophthalmic optics and devices, (170.3890) Medical optics instrumentation     

Two-photon excited hemoglobin fluorescence

We discovered that hemoglobin emits high energy Soret fluorescence when two-photon excited by the visible femtosecond light sources. The unique spectral and temporal characteristics of hemoglobin fluorescence were measured by using a time-resolved spectroscopic detection system. The high energy Soret fluorescence of hemoglobin shows the spectral peak at 438 nm with extremely short lifetime. This discovery enables two-photon excitation fluorescence microscopy to become a potentially powerful tool for in vivo label-free imaging of blood cells and vessels.OCIS codes: (170.0170) Medical optics and biotechnology, (300.6500) spectroscopy, time-resolved, (300.6410) Spectroscopy, multiphoton, (170.2520) Fluorescence microscopy, (180.4315) Nonlinear microscopy     

In vivo imaging of human photoreceptor mosaic with wavefront sensorless adaptive optics optical coherence tomography

Wavefront sensorless adaptive optics optical coherence tomography (WSAO-OCT) is a novel imaging technique for in vivo high-resolution depth-resolved imaging that mitigates some of the challenges encountered with the use of sensor-based adaptive optics designs. This technique replaces the Hartmann Shack wavefront sensor used to measure aberrations with a depth-resolved image-driven optimization algorithm, with the metric based on the OCT volumes acquired in real-time. The custom-built ultrahigh-speed GPU processing platform and fast modal optimization algorithm presented in this paper was essential in enabling real-time, in vivo imaging of human retinas with wavefront sensorless AO correction. WSAO-OCT is especially advantageous for developing a clinical high-resolution retinal imaging system as it enables the use of a compact, low-cost and robust lens-based adaptive optics design. In this report, we describe our WSAO-OCT system for imaging the human photoreceptor mosaic in vivo. We validated our system performance by imaging the retina at several eccentricities, and demonstrated the improvement in photoreceptor visibility with WSAO compensation.OCIS codes: (110.4500) Optical coherence tomography, (010.1080) Active or adaptive optics, (220.1000) Aberration compensation, (170.0110) Imaging systems, (170.4470) Ophthalmology, (120.3890) Medical optics instrumentation     

Retinal adaptive optics imaging with a pyramid wavefront sensor

The pyramid wavefront sensor (P-WFS) has replaced the Shack-Hartmann (SH-) WFS as the sensor of choice for high-performance adaptive optics (AO) systems in astronomy. Many advantages of the P-WFS, such as its adjustable pupil sampling and superior sensitivity, are potentially of great benefit for AO-supported imaging in ophthalmology as well. However, so far no high quality ophthalmic AO imaging was achieved using this novel sensor. Usually, a P-WFS requires modulation and high precision optics that lead to high complexity and costs of the sensor. These factors limit the competitiveness of the P-WFS with respect to other WFS devices for AO correction in visual science. Here, we present a cost-effective realization of AO correction with a non-modulated P-WFS based on standard components and apply this technique to human retinal in vivo imaging using optical coherence tomography (OCT). P-WFS based high quality AO imaging was successfully performed in 5 healthy subjects and smallest retinal cells such as central foveal cone photoreceptors are visualized. The robustness and versatility of the sensor is demonstrated in the model eye under various conditions and in vivo by high-resolution imaging of other structures in the retina using standard and extended fields of view. As a quality benchmark, the performance of conventional SH-WFS based AO was used and successfully met. This work may trigger a paradigm shift with respect to the wavefront sensor of choice for AO in ophthalmic imaging.     

Understanding the changes of cone reflectance in adaptive optics flood illumination retinal images over three years

Although there is increasing interest in the investigation of cone reflectance variability, little is understood about its characteristics over long time scales. Cone detection and its automation is now becoming a fundamental step in the assessment and monitoring of the health of the retina and in the understanding of the photoreceptor physiology. In this work we provide an insight into the cone reflectance variability over time scales ranging from minutes to three years on the same eye, and for large areas of the retina (≥ 2.0 × 2.0 degrees) at two different retinal eccentricities using a commercial adaptive optics (AO) flood illumination retinal camera. We observed that the difference in reflectance observed in the cones increases with the time separation between the data acquisitions and this may have a negative impact on algorithms attempting to track cones over time. In addition, we determined that displacements of the light source within 0.35 mm of the pupil center, which is the farthest location from the pupil center used by operators of the AO camera to acquire high-quality images of the cone mosaic in clinical studies, does not significantly affect the cone detection and density estimation.OCIS codes: (110.1080) Active or adaptive optics, (330.7331) Visual optics, receptor optics, (170.3880) Medical and biological imaging     

Semi-automated identification of cones in the human retina using circle Hough transform

A large number of human retinal diseases are characterized by a progressive loss of cones, the photoreceptors critical for visual acuity and color perception. Adaptive Optics (AO) imaging presents a potential method to study these cells in vivo. However, AO imaging in ophthalmology is a relatively new phenomenon and quantitative analysis of these images remains difficult and tedious using manual methods. This paper illustrates a novel semi-automated quantitative technique enabling registration of AO images to macular landmarks, cone counting and its radius quantification at specified distances from the foveal center. The new cone counting approach employs the circle Hough transform (cHT) and is compared to automated counting methods, as well as arbitrated manual cone identification. We explore the impact of varying the circle detection parameter on the validity of cHT cone counting and discuss the potential role of using this algorithm in detecting both cones and rods separately.OCIS codes: (100.0100) Image processing, (170.3880) Medical and biological imaging, (110.1080) Active or adaptive optics, (170.4470) Ophthalmology     

Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice

Cellular-resolution in vivo fluorescence imaging is a valuable tool for longitudinal studies of retinal function in vision research. Wavefront sensorless adaptive optics (WSAO) is a developing technology that enables high-resolution imaging of the mouse retina. In place of the conventional method of using a Shack-Hartmann wavefront sensor to measure the aberrations directly, WSAO uses an image quality metric and a search algorithm to drive the shape of the adaptive element (i.e. deformable mirror). WSAO is a robust approach to AO and it is compatible with a compact, low-cost lens-based system. In this report, we demonstrated a hill-climbing algorithm for WSAO with a variable focus lens and deformable mirror for non-invasive in vivo imaging of EGFP (enhanced green fluorescent protein) labelled ganglion cells and microglia cells in the mouse retina.OCIS codes: (170.4460) Ophthalmic optics and devices, (010.1080) Active or adaptive optics, (170.0110) Imaging systems, (170.4470) Ophthalmology     

Introduction: Feature Issue on Cellular Imaging of the Retina

The editors introduce the Biomedical Optics Express feature issue, “Cellular Imaging of the Retina,” which includes 14 contributions from the vision and optics community.OCIS codes: (000.1200) Announcements, awards, news, and organizational activities; (110.1080) Active or adaptive optics; (110.2960) Image analysis; (170.0170) Medical optics and biotechnology; (170.4500) Optical coherence tomography; (170.5755) Retina scanning; (330.4460) Ophthalmic optics and devices; (330.5310) Vision-photoreceptors; (330.7327) Visual optics, ophthalmic instrumentationRetinal imaging is a powerful tool, enabling clinicians to directly observe pathology and researchers to probe retinal structure and function. Cellular resolution imaging of the retina is a broad and rapidly growing field, benefiting from continued advances in optical instrumentation as well as data and image processing capabilities. In many cases, this can be accomplished in vivo. Advances in retinal imaging are made possible through interactions between the vision and optics communities, something that the Optical Society of America has staunchly supported throughout its history. Reflecting the continued commitment to this field, we were invited by the editors at Biomedical Optics Express to organize a feature issue highlighting recent advances in the field of Cellular Imaging of the Retina. We were quite pleased with the response (outlined below), and believe the assembled feature issue appropriately captures the current state of the field.The 14 papers in this issue span many disciplines, including clinical imaging, new instrumentation, image analysis, imaging in animal models, and functional retinal imaging. Reflecting the multidisciplinary nature of this field, most papers fit in multiple categories, and here we provide a brief précis of the entire issue.Using a variety of imaging techniques, the majority of the papers examined the human retina. Among the imaging tools used to acquire retinal images were stand-alone and adaptive-optics enhanced: scanning laser ophthalmoscopy/scanning ophthalmoscopy (AOSLO/AOSO), optical coherence tomography (OCT), and fundus imaging. Adaptive optics (AO) has opened numerous avenues of research in cellular resolution retinal imaging, thus it is no surprise that we find AO at the heart of many of the papers in this feature issue.Dubra et al. [1], Zawadzki et al. [2], Manzanera et al. [3], and Vohnsen and Rativa [4] introduced new instrumentation for imaging the photoreceptor mosaic using a broadband AOSO, an integrated AO-OCT SLO, a new MEMS-based AOSLO, and an annular-illumination SLO, respectively. What we take away from these papers is that there is a continued drive to improve the technology even further, which must occur in parallel to robust application of existing imaging tools to studying the retina in order to keep the field moving forward at its current pace.Four additional papers examined structural and functional properties of the photoreceptor mosaic. Kacaoglu et al. [5] examined spatial and temporal evaluation of individual cone photoreceptors using AO-OCT. Rativa and Vohnsen [6] probed the directionality of cone photoreceptors at different retinal eccentricities using scanning laser ophthalmoscopy. Dees et al. [7] examined variability in parafoveal cone density in normal trichromatic individuals using a high-speed flood-illuminated AO fundus camera. Dubra et al. [8] move beyond cones and demonstrate striking images of the rod photoreceptor mosaic at various retinal locations using a newly developed confocal AOSO.Moving to clinical imaging, Tam et al. [9] developed a method to characterize capillary flow dynamics in the living human retina using an AOSLO, something which could be quite valuable for studying certain retinal diseases. In glaucoma patients, Hood and Raza [10] provide a method for comparing functional visual field defects to local nerve fiber and retinal ganglion cell damage seen on OCT. Hood et al. [11] present a method to define visual field boundaries from an analysis of the inner segment/outer segment border in OCT images of patients with retinitis pigmentosa. While the image resolution in these latter cases is not cellular, the approach provides a key step forward in linking retinal structure with function, and could be extended to some of the cellular imaging techniques described in this feature issue.The final three papers applied high-resolution imaging techniques to animal models. Moayed et al. [12] provide volumetric imaging of the chicken retina in vivo using spectral domain OCT. Also in the chicken, but ex vivo, Bueno et al. [13] developed an AO multiphoton microscope for examining multiple retinal layers. Lu et al. [14] examined the cellular sources of autofluorescence in freshly isolated frog retinas using a two-photon excitation fluorescence microscope. Ultimately, there are questions that just can’t be addressed by imaging the human retina, and animal models will continue to be needed. As such, adaptation and development of cellular imaging systems for use in various animal preparations remains an area of need in this field.All papers in this issue have undergone a rigorous peer review process, and we are indebted to the referees for their efforts in ensuring that the Optical Society of America’s standards for quality and integrity were met. We are especially gratefully to Joseph A. Izatt (Editor-in-Chief), Gregory W. Faris (Deputy Editor), and the publication staff at the Optical Society of America for their hard work and dedication to this feature issue: Joe Richardson, Miriam Day, Kelly Cohen and the many others who contributed behind the scenes. We hope you find the papers in this feature issue as enlightening as we did, and expect that they will stimulate research to further move the field forward.     

Visualizing retinal cells with adaptive optics imaging modalities using a translational imaging framework

Adaptive optics reflectance-based retinal imaging has proved a valuable tool for the noninvasive visualization of cells in the living human retina. Many subcellular features that remain at or below the resolution limit of current in vivo techniques may be more easily visualized with the same modalities in an ex vivo setting. While most microscopy techniques provide significantly higher resolution, enabling the visualization of fine cellular detail in ex vivo retinal samples, they do not replicate the reflectance-based imaging modalities of in vivo retinal imaging. Here, we introduce a strategy for imaging ex vivo samples using the same imaging modalities as those used for in vivo retinal imaging, but with increased resolution. We also demonstrate the ability of this approach to perform protein-specific fluorescence imaging and reflectance imaging simultaneously, enabling the visualization of nearly transparent layers of the retina and the classification of cone photoreceptor types.     

Adaptive optics optical coherence tomography with dynamic retinal tracking

Adaptive optics optical coherence tomography (AO-OCT) is a highly sensitive and noninvasive method for three dimensional imaging of the microscopic retina. Like all in vivo retinal imaging techniques, however, it suffers the effects of involuntary eye movements that occur even under normal fixation. In this study we investigated dynamic retinal tracking to measure and correct eye motion at KHz rates for AO-OCT imaging. A customized retina tracking module was integrated into the sample arm of the 2nd-generation Indiana AO-OCT system and images were acquired on three subjects. Analyses were developed based on temporal amplitude and spatial power spectra in conjunction with strip-wise registration to independently measure AO-OCT tracking performance. After optimization of the tracker parameters, the system was found to correct eye movements up to 100 Hz and reduce residual motion to 10 µm root mean square. Between session precision was 33 µm. Performance was limited by tracker-generated noise at high temporal frequencies.OCIS codes: (110.1080) Active or adaptive optics, (170.4500) Optical coherence tomography, (120.3890) Medical optics instrumentation, (170.0110) Imaging systems, (170.4470) Ophthalmology, (330.5310) Vision - photoreceptors     

Reflective mirror-based line-scan adaptive optics OCT for imaging retinal structure and function

Line-scan OCT incorporated with adaptive optics (AO) offers high resolution, speed, and sensitivity for imaging retinal structure and function in vivo. Here, we introduce its implementation with reflective mirror-based afocal telescopes, optimized for imaging light-induced retinal activity (optoretinography) and weak retinal reflections at the cellular scale. A non-planar optical design was followed based on previous recommendations with key differences specific to a line-scan geometry. The three beam paths fundamental to an OCT system –illumination/sample, detection, and reference– were modeled in Zemax optical design software to yield theoretically diffraction-limited performance over a 2.2 deg. field-of-view and 1.5 D vergence range at the eye’s pupil. The performance for imaging retinal structure was exemplified by cellular-scale visualization of retinal ganglion cells, macrophages, foveal cones, and rods in human observers. The performance for functional imaging was exemplified by resolving the light-evoked optical changes in foveal cone photoreceptors where the spatial resolution was sufficient for cone spectral classification at an eccentricity 0.3 deg. from the foveal center. This enabled the first in vivo demonstration of reduced S-cone (short-wavelength cone) density in the human foveola, thus far observed only in ex vivo histological preparations. Together, the feasibility for high resolution imaging of retinal structure and function demonstrated here holds significant potential for basic science and translational applications.     

Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope

The rod photoreceptors are implicated in a number of devastating retinal diseases. However, routine imaging of these cells has remained elusive, even with the advent of adaptive optics imaging. Here, we present the first in vivo images of the contiguous rod photoreceptor mosaic in nine healthy human subjects. The images were collected with three different confocal adaptive optics scanning ophthalmoscopes at two different institutions, using 680 and 775 nm superluminescent diodes for illumination. Estimates of photoreceptor density and rod:cone ratios in the 5°-15° retinal eccentricity range are consistent with histological findings, confirming our ability to resolve the rod mosaic by averaging multiple registered images, without the need for additional image processing. In one subject, we were able to identify the emergence of the first rods at approximately 190 μm from the foveal center, in agreement with previous histological studies. The rod and cone photoreceptor mosaics appear in focus at different retinal depths, with the rod mosaic best focus (i.e., brightest and sharpest) being at least 10 μm shallower than the cones at retinal eccentricities larger than 8°. This study represents an important step in bringing high-resolution imaging to bear on the study of rod disorders.     

Tracking features in retinal images of adaptive optics confocal scanning laser ophthalmoscope using KLT-SIFT algorithm

With the use of adaptive optics (AO), high-resolution microscopic imaging of living human retina in the single cell level has been achieved. In an adaptive optics confocal scanning laser ophthalmoscope (AOSLO) system, with a small field size (about 1 degree, 280 μm), the motion of the eye severely affects the stabilization of the real-time video images and results in significant distortions of the retina images. In this paper, Scale-Invariant Feature Transform (SIFT) is used to abstract stable point features from the retina images. Kanade-Lucas-Tomasi(KLT) algorithm is applied to track the features. With the tracked features, the image distortion in each frame is removed by the second-order polynomial transformation, and 10 successive frames are co-added to enhance the image quality. Features of special interest in an image can also be selected manually and tracked by KLT. A point on a cone is selected manually, and the cone is tracked from frame to frame.