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.OCIS codes: (010.1080) adaptive optics, (330.4460) Ophthalmic optics and devices, (330.5310) Vision – photoreceptors, (330.7327) Visual optics, ophthalmic instrumentation     

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.     

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.     

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     

Wavefront sensorless adaptive optics optical coherence tomography for in vivo retinal imaging in mice

We present wavefront sensorless adaptive optics (WSAO) Fourier domain optical coherence tomography (FD-OCT) for in vivo small animal retinal imaging. WSAO is attractive especially for mouse retinal imaging because it simplifies optical design and eliminates the need for wavefront sensing, which is difficult in the small animal eye. GPU accelerated processing of the OCT data permitted real-time extraction of image quality metrics (intensity) for arbitrarily selected retinal layers to be optimized. Modal control of a commercially available segmented deformable mirror (IrisAO Inc.) provided rapid convergence using a sequential search algorithm. Image quality improvements with WSAO OCT are presented for both pigmented and albino mouse retinal data, acquired in vivo.OCIS codes: (170.4460) Ophthalmic optics and devices, (110.1080) Active or adaptive optics, (110.4500) Optical coherence tomography     

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     

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 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     

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     

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     

A two-photon lysosome-targeted probe for endogenous formaldehyde in living cells

Formaldehyde (FA) is a gaseous signaling molecule that plays a vital role in various biological processes as well as neurodegenerative diseases. Therefore, it is of great practical significance to develop effective and reliable chemical sensors for the monitoring of endogenous FA. Here, we designed and synthesized a two-photon (810 nm) turn-on chemosensor AMNT (aminomorpholine naphthalimide) that accurately localizes lysosomes in cells for imaging of cellular endogenous FA. The fluorescence emission peak of AMNT was at ∼540 nm, with a slight blue shift (∼528 nm) in response to FA, while the green fluorescence intensity increased. The probe exhibits excellent selectivity for FA among other biological interference species and a fast response time for FA. It is worth mentioning that the probe successfully imaged endogenous FA in cells in two-photon mode, making the probe an effective research tool in the biomedical field to study diseases related to abnormal FA expression.

A turn-on two-photon lysosome-targeted probe based on the ICT mechanism has been synthesized and was successfully used not only to monitor and image formaldehyde exogenously but also endogenously with excellent performance in living cells.     

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     

Long-term two-photon neuroimaging with a photostable AIE luminogen

In neuroscience, fluorescence labeled two-photon microscopy is a promising tool to visualize ex vivo and in vivo tissue morphology, and track dynamic neural activities. Specific and highly photostable fluorescent probes are required in this technology. However, most fluorescent proteins and organic fluorophores suffer from photobleaching, so they are not suitable for long-term imaging and observation. To overcome this problem, we utilize tetraphenylethene-triphenylphosphonium (TPE-TPP), which possesses aggregation-induced emission (AIE) and two-photon fluorescence characteristics, for neuroimaging. The unique AIE feature of TPE-TPP makes its nanoaggregates resistant to photobleaching, which is useful to track neural cells and brain-microglia for a long period of time. Two-photon fluorescence of TPE-TPP facilitates its application in deep in vivo neuroimaging, as demonstrated in the present paper.OCIS codes: (160.4890) Organic materials, (160.2540) Fluorescent and luminescent materials, (190.4180) Multiphoton processes, (170.3880) Medical and biological imaging, (180.2520) Fluorescence microscopy, (180.4315) Nonlinear microscopy     

Retinal, anterior segment and full eye imaging using ultrahigh speed swept source OCT with vertical-cavity surface emitting lasers

We demonstrate swept source OCT utilizing vertical-cavity surface emitting laser (VCSEL) technology for in vivo high speed retinal, anterior segment and full eye imaging. The MEMS tunable VCSEL enables long coherence length, adjustable spectral sweep range and adjustable high sweeping rate (50–580 kHz axial scan rate). These features enable integration of multiple ophthalmic applications into one instrument. The operating modes of the device include: ultrahigh speed, high resolution retinal imaging (up to 580 kHz); high speed, long depth range anterior segment imaging (100 kHz) and ultralong range full eye imaging (50 kHz). High speed imaging enables wide-field retinal scanning, while increased light penetration at 1060 nm enables visualization of choroidal vasculature. Comprehensive volumetric data sets of the anterior segment from the cornea to posterior crystalline lens surface are also shown. The adjustable VCSEL sweep range and rate make it possible to achieve an extremely long imaging depth range of ~50 mm, and to demonstrate the first in vivo 3D OCT imaging spanning the entire eye for non-contact measurement of intraocular distances including axial eye length. Swept source OCT with VCSEL technology may be attractive for next generation integrated ophthalmic OCT instruments.OCIS codes: (110.4500) Optical coherence tomography, (120.4640) Optical instruments, (140.3600) Lasers, tunable, (170.4460) Ophthalmic optics and devices, (170.4470) Ophthalmology     

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.     

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     

Improved two-photon imaging of living neurons in brain tissue through temporal gating

We optimize two-photon imaging of living neurons in brain tissue by temporally gating an incident laser to reduce the photon flux while optimizing the maximum fluorescence signal from the acquired images. Temporal gating produces a bunch of ~10 femtosecond pulses and the fluorescence signal is improved by increasing the bunch-pulse energy. Gating is achieved using an acousto-optic modulator with a variable gating frequency determined as integral multiples of the imaging sampling frequency. We hypothesize that reducing the photon flux minimizes the photo-damage to the cells. Our results, however, show that despite producing a high fluorescence signal, cell viability is compromised when the gating and sampling frequencies are equal (or effectively one bunch-pulse per pixel). We found an optimum gating frequency range that maintains the viability of the cells while preserving a pre-set fluorescence signal of the acquired two-photon images. The neurons are imaged while under whole-cell patch, and the cell viability is monitored as a change in the membrane’s input resistance.OCIS codes: (170.3880) Medical and biological imaging, (170.3660) Light propagation in tissues, (140.0140) Lasers and laser optics, (170.2520) Fluorescence microscopy, (170.6930) Tissue, (110.0110) Imaging systems     

Dual-modality fiber-based OCT-TPL imaging system for simultaneous microstructural and molecular analysis of atherosclerotic plaques

New optical imaging techniques that provide contrast to study both the anatomy and composition of atherosclerotic plaques can be utilized to better understand the formation, progression and clinical complications of human coronary artery disease. We present a dual-modality fiber-based optical imaging system for simultaneous microstructural and molecular analysis of atherosclerotic plaques that combines optical coherence tomography (OCT) and two-photon luminescence (TPL) imaging. Experimental results from ex vivo human coronary arteries show that OCT and TPL optical contrast in recorded OCT-TPL images is complimentary and in agreement with histological analysis. Molecular composition (e.g., lipid and oxidized-LDL) detected by TPL imaging can be overlaid onto plaque microstructure depicted by OCT, providing new opportunities for atherosclerotic plaque identification and characterization.OCIS codes: (110.4500) Optical coherence tomography, (190.1900) Diagnostic applications of nonlinear optics, (190.4370) Nonlinear optics, fibers, (170.6280) Spectroscopy, fluorescence and luminescence, (170.6935) Tissue characterization     

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.     

The use of forward scatter to improve retinal vascular imaging with an adaptive optics scanning laser ophthalmoscope

Retinal vascular diseases are a leading cause of blindness and visual disability. The advent of adaptive optics retinal imaging has enabled us to image the retinal vascular at cellular resolutions, but imaging of the vasculature can be difficult due to the complex nature of the images, including features of many other retinal structures, such as the nerve fiber layer, glial and other cells. In this paper we show that varying the size and centration of the confocal aperture of an adaptive optics scanning laser ophthalmoscope (AOSLO) can increase sensitivity to multiply scattered light, especially light forward scattered from the vasculature and erythrocytes. The resulting technique was tested by imaging regions with different retinal tissue reflectivities as well as within the optic nerve head.OCIS codes: (110.1085) Adaptive imaging, (110.1220) Apertures, (170.4460) Ophthalmic optics and devices, (170.1470) Blood or tissue constituent monitoring