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     

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     

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.     

Contrast-based sensorless adaptive optics for retinal imaging

Conventional adaptive optics ophthalmoscopes use wavefront sensing methods to characterize ocular aberrations for real-time correction. However, there are important situations in which the wavefront sensing step is susceptible to difficulties that affect the accuracy of the correction. To circumvent these, wavefront sensorless adaptive optics (or non-wavefront sensing AO; NS-AO) imaging has recently been developed and has been applied to point-scanning based retinal imaging modalities. In this study we show, for the first time, contrast-based NS-AO ophthalmoscopy for full-frame in vivo imaging of human and animal eyes. We suggest a robust image quality metric that could be used for any imaging modality, and test its performance against other metrics using (physical) model eyes.OCIS codes: (010.1080) Active or adaptive optics, (170.3880) Medical and biological imaging, (170.4460) Ophthalmic optics and devices, (330.4875) Optics of physiological systems     

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     

Extended depth of focus adaptive optics spectral domain optical coherence tomography

We present an adaptive optics spectral domain optical coherence tomography (AO-SDOCT) with a long focal range by active phase modulation of the pupil. A long focal range is achieved by introducing AO-controlled third-order spherical aberration (SA). The property of SA and its effects on focal range are investigated in detail using the Huygens-Fresnel principle, beam profile measurement and OCT imaging of a phantom. The results indicate that the focal range is extended by applying SA, and the direction of extension can be controlled by the sign of applied SA. Finally, we demonstrated in vivo human retinal imaging by altering the applied SA.OCIS codes: (170.4500) Optical coherence tomography, (110.1080) Active or adaptive optics, (170.4470) Ophthalmology     

Computed optical interferometric tomography for high-speed volumetric cellular imaging

Three-dimensional high-resolution imaging methods are important for cellular-level research. Optical coherence microscopy (OCM) is a low-coherence-based interferometry technology for cellular imaging with both high axial and lateral resolution. Using a high-numerical-aperture objective, OCM normally has a shallow depth of field and requires scanning the focus through the entire region of interest to perform volumetric imaging. With a higher-numerical-aperture objective, the image quality of OCM is affected by and more sensitive to aberrations. Interferometric synthetic aperture microscopy (ISAM) and computational adaptive optics (CAO) are computed imaging techniques that overcome the depth-of-field limitation and the effect of optical aberrations in optical coherence tomography (OCT), respectively. In this work we combine OCM with ISAM and CAO to achieve high-speed volumetric cellular imaging. Experimental imaging results of ex vivo human breast tissue, ex vivo mouse brain tissue, in vitro fibroblast cells in 3D scaffolds, and in vivo human skin demonstrate the significant potential of this technique for high-speed volumetric cellular imaging.OCIS codes: (170.4500) Optical coherence tomography, (090.1000) Aberration compensation, (180.3170) Interference microscopy, (170.6900) Three-dimensional microscopy, (110.1758) Computational imaging, (100.3200) Inverse scattering     

Integrated adaptive optics optical coherence tomography and adaptive optics scanning laser ophthalmoscope system for simultaneous cellular resolution in vivo retinal imaging

We describe an ultrahigh-resolution (UHR) retinal imaging system that combines adaptive optics Fourier-domain optical coherence tomography (AO-OCT) with an adaptive optics scanning laser ophthalmoscope (AO-SLO) to allow simultaneous data acquisition by the two modalities. The AO-SLO subsystem was integrated into the previously described AO-UHR OCT instrument with minimal changes to the latter. This was done in order to ensure optimal performance and image quality of the AO- UHR OCT. In this design both imaging modalities share most of the optical components including a common AO-subsystem and vertical scanner. One of the benefits of combining Fd-OCT with SLO includes automatic co-registration between two acquisition channels for direct comparison between retinal structures imaged by both modalities (e.g., photoreceptor mosaics or microvasculature maps). Because of differences in the detection scheme of the two systems, this dual imaging modality instrument can provide insight into retinal morphology and potentially function, that could not be accessed easily by a single system. In this paper we describe details of the components and parameters of the combined instrument, including incorporation of a novel membrane magnetic deformable mirror with increased stroke and actuator count used as a single wavefront corrector. We also discuss laser safety calculations for this multimodal system. Finally, retinal images acquired in vivo with this system are presented.     

Heartbeat OCT: in vivo intravascular megahertz-optical coherence tomography

Cardiac motion artifacts, non-uniform rotational distortion and undersampling affect the image quality and the diagnostic impact of intravascular optical coherence tomography (IV-OCT). In this study we demonstrate how these limitations of IV-OCT can be addressed by using an imaging system that we called “Heartbeat OCT”, combining a fast Fourier Domain Mode Locked laser, fast pullback, and a micromotor actuated catheter, designed to examine a coronary vessel in less than one cardiac cycle. We acquired in vivo data sets of two coronary arteries in a porcine heart with both Heartbeat OCT, working at 2.88 MHz A-line rate, 4000 frames/s and 100 mm/s pullback speed, and with a commercial system. The in vivo results show that Heartbeat OCT provides faithfully rendered, motion-artifact free, fully sampled vessel wall architecture, unlike the conventional IV-OCT data. We present the Heartbeat OCT system in full technical detail and discuss the steps needed for clinical translation of the technology.OCIS codes: (170.4500) Optical coherence tomography, (170.3880) Medical and biological imaging, (170.2150) Endoscopic imaging, (110.6880) Three-dimensional image acquisition, (110.2350) Fiber optics imaging, (120.5800) Scanners, (120.3890) Medical optics instrumentation     

Closed-loop wavefront sensing and correction in the mouse brain with computed optical coherence microscopy

Optical coherence microscopy (OCM) uses interferometric detection to capture the complex optical field with high sensitivity, which enables computational wavefront retrieval using back-scattered light from the sample. Compared to a conventional wavefront sensor, aberration sensing with OCM via computational adaptive optics (CAO) leverages coherence and confocal gating to obtain signals from the focus with less cross-talk from other depths or transverse locations within the field-of-view. Here, we present an investigation of the performance of CAO-based aberration sensing in simulation, bead phantoms, and ex vivo mouse brain tissue. We demonstrate that, due to the influence of the double-pass confocal OCM imaging geometry on the shape of computed pupil functions, computational sensing of high-order aberrations can suffer from signal attenuation in certain spatial-frequency bands and shape similarity with lower order counterparts. However, by sensing and correcting only low-order aberrations (astigmatism, coma, and trefoil), we still successfully corrected tissue-induced aberrations, leading to 3× increase in OCM signal intensity at a depth of ∼0.9 mm in a freshly dissected ex vivo mouse brain.     

A high-efficiency fiber-based imaging system for co-registered autofluorescence and optical coherence tomography

We present a power-efficient fiber-based imaging system capable of co-registered autofluorescence imaging and optical coherence tomography (AF/OCT). The system employs a custom fiber optic rotary joint (FORJ) with an embedded dichroic mirror to efficiently combine the OCT and AF pathways. This three-port wavelength multiplexing FORJ setup has a throughput of more than 83% for collected AF emission, significantly more efficient compared to previously reported fiber-based methods. A custom 900 µm diameter catheter ‒ consisting of a rotating lens assembly, double-clad fiber (DCF), and torque cable in a stationary plastic tube ‒ was fabricated to allow AF/OCT imaging of small airways in vivo. We demonstrate the performance of this system ex vivo in resected porcine airway specimens and in vivo in human on fingers, in the oral cavity, and in peripheral airways.OCIS codes: (110.0110) Imaging systems, (110.2350) Fiber optics imaging, (110.4500) Optical coherence tomography, (170.2520) Fluorescence microscopy, (170.3890) Medical optics 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     

Handheld simultaneous scanning laser ophthalmoscopy and optical coherence tomography system

Scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) are widely used retinal imaging modalities that can assist in the diagnosis of retinal pathologies. The combination of SLO and OCT provides a more comprehensive imaging system and a method to register OCT images to produce motion corrected retinal volumes. While high quality, bench-top SLO-OCT systems have been discussed in the literature and are available commercially, there are currently no handheld designs. We describe the first design and fabrication of a handheld SLO/spectral domain OCT probe. SLO and OCT images were acquired simultaneously with a combined power under the ANSI limit. High signal-to-noise ratio SLO and OCT images were acquired simultaneously from a normal subject with visible motion artifacts. Fully automated motion estimation methods were performed in post-processing to correct for the inter- and intra-frame motion in SLO images and their concurrently acquired OCT volumes. The resulting set of reconstructed SLO images and the OCT volume were without visible motion artifacts. At a reduced field of view, the SLO resolved parafoveal cones without adaptive optics at a retinal eccentricity of 11° in subjects with good ocular optics. This system may be especially useful for imaging young children and subjects with less stable fixation.OCIS codes: (170.4460) Ophthalmic optics and devices, (080.3620) Lens system design, (170.0110) Imaging systems, (170.5755) Retina scanning, (170.4470) Ophthalmology, (110.4500) Optical coherence tomography, (110.4153) Motion estimation and optical flow     

In vivo imaging of retinal hemodynamics with OCT angiography and Doppler OCT

Retinal hemodynamics is important for early diagnosis and precise monitoring in retinal vascular diseases. We propose a novel method for measuring absolute retinal blood flow in vivo using the combined techniques of optical coherence tomography (OCT) angiography and Doppler OCT. Doppler values can be corrected by Doppler angles extracted from OCT angiography images. A three-dimensional (3D) segmentation algorithm based on dynamic programming was developed to extract the 3D boundaries of optic disc vessels, and Doppler angles were calculated from 3D vessel geometry. The accuracy of blood flow from the Doppler OCT was validated using a flow phantom. The feasibility of the method was tested on a subject in vivo. The pulsatile retinal blood flow and the parameters for retinal hemodynamics were successfully obtained.OCIS codes: (100.2960) Image analysis, (100.6890) Three-dimensional image processing, (170.4500) Optical coherence tomography, (170.4460) Ophthalmic optics and devices     

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.     

Imaging of retinal vasculature using adaptive optics SLO/OCT

We use our previously developed adaptive optics (AO) scanning laser ophthalmoscope (SLO)/ optical coherence tomography (OCT) instrument to investigate its capability for imaging retinal vasculature. The system records SLO and OCT images simultaneously with a pixel to pixel correspondence which allows a direct comparison between those imaging modalities. Different field of views ranging from 0.8°x0.8° up to 4°x4° are supported by the instrument. In addition a dynamic focus scheme was developed for the AO-SLO/OCT system in order to maintain the high transverse resolution throughout imaging depth. The active axial eye tracking that is implemented in the OCT channel allows time resolved measurements of the retinal vasculature in the en-face imaging plane. Vessel walls and structures that we believe correspond to individual erythrocytes could be visualized with the system.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     

Photothermal optical lock-in optical coherence tomography for in vivo imaging

Photothermal OCT (PTOCT) provides high sensitivity to molecular targets in tissue, and occupies a spatial imaging regime that is attractive for small animal imaging. However, current implementations of PTOCT require extensive temporal sampling, resulting in slow frame rates and a large data burden that limit its in vivo utility. To address these limitations, we have implemented optical lock-in techniques for photothermal optical lock-in OCT (poli-OCT), and demonstrated the in vivo imaging capabilities of this approach. The poli-OCT signal was assessed in tissue-mimicking phantoms containing indocyanine green (ICG), an FDA approved small molecule that has not been previously imaged in vivo with PTOCT. Then, the effects of in vivo blood flow and motion artifact were assessed and attenuated, and in vivo poli-OCT was demonstrated with both ICG and gold nanorods as contrast agents. Experiments revealed that poli-OCT signals agreed with optical lock-in theory and the bio-heat equation, and the system exhibited shot noise limited performance. In phantoms containing biologically relevant concentrations of ICG (1 µg/ml), the poli-OCT signal was significantly greater than control phantoms (p<0.05), demonstrating sensitivity to small molecules. Finally, in vivo poli-OCT of ICG identified the lymphatic vessels in a mouse ear, and also identified low concentrations (200 pM) of gold nanorods in subcutaneous injections at frame rates ten times faster than previously reported. This work illustrates that future in vivo molecular imaging studies could benefit from the improved acquisition and analysis times enabled by poli-OCT.OCIS codes: (110.4500) Optical coherence tomography, (350.5340) Photothermal effects     

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     

Dual instrument for in vivo and ex vivo OCT imaging in an ENT department

A dual instrument is assembled to investigate the usefulness of optical coherence tomography (OCT) imaging in an ear, nose and throat (ENT) department. Instrument 1 is dedicated to in vivo laryngeal investigation, based on an endoscope probe head assembled by compounding a miniature transversal flying spot scanning probe with a commercial fiber bundle endoscope. This dual probe head is used to implement a dual channel nasolaryngeal endoscopy-OCT system. The two probe heads are used to provide simultaneously OCT cross section images and en face fiber bundle endoscopic images. Instrument 2 is dedicated to either in vivo imaging of accessible surface skin and mucosal lesions of the scalp, face, neck and oral cavity or ex vivo imaging of the same excised tissues, based on a single OCT channel. This uses a better interface optics in a hand held probe. The two instruments share sequentially, the swept source at 1300 nm, the photo-detector unit and the imaging PC. An aiming red laser is permanently connected to the two instruments. This projects visible light collinearly with the 1300 nm beam and allows pixel correspondence between the en face endoscopy image and the cross section OCT image in Instrument 1, as well as surface guidance in Instrument 2 for the operator. The dual channel instrument was initially tested on phantom models and then on patients with suspect laryngeal lesions in a busy ENT practice. This feasibility study demonstrates the OCT potential of the dual imaging instrument as a useful tool in the testing and translation of OCT technology from the lab to the clinic. Instrument 1 is under investigation as a possible endoscopic screening tool for early laryngeal cancer. Larger size and better quality cross-section OCT images produced by Instrument 2 provide a reference base for comparison and continuing research on imaging freshly excised tissue, as well as in vivo interrogation of more superficial skin and mucosal lesions in the head and neck patient.OCIS codes: (170.4500) Optical coherence tomography, (170.2150) Endoscopic imaging, (170.3890) Medical optics instrumentation, (170.1610) Clinical applications     

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