Daniel J. Wahl

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.

Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging.

Multiphoton microscopy enables imaging deep into scattering tissues. The efficient generation of non-linear optical effects is related to both the pulse duration (typically on the order of femtoseconds) and the size of the focused spot. Aberrations introduced by refractive index inhomogeneity in the sample distort the wavefront and enlarge the focal spot, which reduces the multiphoton signal. Traditional approaches to adaptive optics wavefront correction are not effective in thick or multi-layered scattering media. In this report, we present sensorless adaptive optics (SAO) using low-coherence interferometric detection of the excitation light for depth-resolved aberration correction of two-photon excited fluorescence (TPEF) in biological tissue. We demonstrate coherence-gated SAO TPEF using a transmissive multi-actuator adaptive lens for in vivo imaging in a mouse retina. This configuration has significant potential for reducing the laser power required for adaptive optics multiphoton imaging, and for facilitating integration with existing systems.

Wavefront sensorless adaptive optics OCT with the DONE algorithm for in vivo human retinal imaging [Invited]

In this report, which is an international collaboration of OCT, adaptive optics, and control research, we demonstrate the Data-based Online Nonlinear Extremum-seeker (DONE) algorithm to guide the image based optimization for wavefront sensorless adaptive optics (WFSL-AO) OCT for in vivo human retinal imaging. The ocular aberrations were corrected using a multi-actuator adaptive lens after linearization of the hysteresis in the piezoelectric actuators. The DONE algorithm succeeded in drastically improving image quality and the OCT signal intensity, up to a factor seven, while achieving a computational time of 1 ms per iteration, making it applicable for many high speed applications. We demonstrate the correction of five aberrations using 70 iterations of the DONE algorithm performed over 2.8 s of continuous volumetric OCT acquisition. Data acquired from an imaging phantom and in vivo from human research volunteers are presented.

Multiscale sensorless adaptive optics OCT angiography system for in vivo human retinal imaging

Abstract. We present a multiscale sensorless adaptive optics (SAO) OCT system capable of imaging retinal structure and vasculature with various fields-of-view (FOV) and resolutions. Using a single deformable mirror and exploiting the polarization properties of light, the SAO-OCT-A was implemented in a compact and easy to operate system. With the ability to adjust the beam diameter at the pupil, retinal imaging was demonstrated at two different numerical apertures with the same system. The general morphological structure and retinal vasculature could be observed with a few tens of micrometer-scale lateral resolution with conventional OCT and OCT-A scanning protocols with a 1.7-mm-diameter beam incident at the pupil and a large FOV (15 deg× 15 deg). Changing the system to a higher numerical aperture with a 5.0-mm-diameter beam incident at the pupil and the SAO aberration correction, the FOV was reduced to 3 deg× 3 deg for fine detailed imaging of morphological structure and microvasculature such as the photoreceptor mosaic and capillaries. Multiscale functional SAO-OCT imaging was performed on four healthy subjects, demonstrating its functionality and potential for clinical utility.

Localization and functional characterization of the p.Asn965Ser (N965S) ABCA4 variant in mice reveal pathogenic mechanisms underlying Stargardt macular degeneration

ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) proteins that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across outer segment disc membranes thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptor cells. Mutations in the gene encoding ABCA4 are responsible for Stargardt disease (STGD1), an autosomal recessive retinal degenerative disease that causes severe vision loss. To define the molecular basis for STGD1 associated with the p.Asn965Ser (N965S) mutation in the Walker A motif of nucleotide binding domain 1 (NBD1), we generated a p.Asn965Ser knockin mouse and compared the subcellular localization and molecular properties of the disease variant with wild-type (WT) ABCA4. Here, we show that the p.Asn965Ser ABCA4 variant expresses at half the level of WT ABCA4, partially mislocalizes to the endoplasmic reticulum (ER) of photoreceptors, is devoid of N-Ret-PE activated ATPase activity, and causes an increase in autofluorescence and the bisretinoid A2E associated with lipofuscin deposits in retinal pigment epithelial cells as found in Stargardt patients and Abca4 knockout mice. We also show for the first time that a significant fraction of WT ABCA4 is retained in the inner segment of photoreceptors. On the basis of these studies we conclude that loss in substrate-dependent ATPase activity and protein misfolding are mechanisms underlying STGD1 associated with the p.Asn965Ser mutation in ABCA4. Functional and molecular modeling studies further suggest that similar pathogenic mechanisms are responsible for Tangiers disease associated with the p.Asn935Ser (N935S) mutation in the NBD1 Walker A motif of ABCA1.

Pupil segmentation adaptive optics for invivo mouse retinal fluorescence imaging

Adaptive Optics (AO) for scanning laser ophthalmoscopy enables high-resolution retinal imaging that can be used for preclinical research of diseases causing vision loss. Pupil Segmentation (PS) is an approach to wavefront-sensorless AO that acquires images within subregions across the imaging pupil to measure the wavefront slopes at the corresponding locations of the beam. We present PS-AO as an approach to correct ocular aberrations in ∼7  s, implemented to minimize respiratory motion from an anesthetized mouse. We demonstrated an improvement in resolution and an image intensity increase of ∼25% across all results using PS-AO for in vivo fluorescence retinal imaging in mice using a MEMS-based segmented deformable mirror.

Wavefront sensorless approaches to adaptive optics for in vivo fluorescence imaging of mouse retina

Adaptive optics (AO) is necessary to correct aberrations when imaging the mouse eye with high numerical aperture. In order to obtain cellular resolution, we have implemented wavefront sensorless adaptive optics for in vivo fluorescence imaging of mouse retina. Our approach includes a lens-based system and MEMS deformable mirror for aberration correction. The AO system was constructed with a reflectance channel for structural images and fluorescence channel for functional images. The structural imaging was used in real-time for navigation on the retina using landmarks such as blood vessels. We have also implemented a tunable liquid lens to select the retinal layer of interest at which to perform the optimization. At the desired location on the mouse retina, the optimization algorithm used the fluorescence image data to drive a modal hill-climbing algorithm using an intensity or sharpness image quality metric. The optimization requires ~30 seconds to complete a search up to the 20th Zernike mode. In this report, we have demonstrated the AO performance for high-resolution images of the capillaries in a fluorescence angiography. We have also made progress on an approach to AO with pupil segmentation as a possible sensorless technique suitable for small animal retinal imaging. Pupil segmentation AO was implemented on the same ophthalmic system and imaging performance was demonstrated on fluorescent beads with induced aberrations.

Wavefront Sensoless Adaptive Optics for Ophthalmic Imaging

Wavefront sensorless adaptive optics is a novel technique that facilitates high resolution ophthalmic imaging such as and SLO; it replaces the Hartmann Shack wavefront sensor with an image-driven optimization algorithm and mitigates some challenges encountered with sensor-based designs.

Adaptive optics with combined optical coherence tomography and scanning laser ophthalmoscopy for in vivo mouse retina imaging

Optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) are two state-of-the-art imaging technologies commonly used to study retina. Adaptive Optics (AO) methodologies enable high-fidelity correction of ocular aberrations, resulting in improved resolution and sensitivity for both SLO and OCT systems. Here we present work integrating OCT into a previously described mouse retinal AO-SLO system, allowing simultaneous reflectance and fluorescence imaging. The new system allows simultaneous data acquisition of AO-SLO and AO-OCT, facilitating registration and comparison of data from both modalities. The system has data acquisition speed of 200 kHz A-scans/pixel, and high volumetric resolution.

Effect of a contact lens on mouse retinal in vivo imaging: Effective focal length changes and monochromatic aberrations

ABSTRACT For in vivo mouse retinal imaging, especially with Adaptive Optics instruments, application of a contact lens is desirable, as it allows maintenance of cornea hydration and helps to prevent cataract formation during lengthy imaging sessions. However, since the refractive elements of the eye (cornea and lens) serve as the objective for most in vivo retinal imaging systems, the use of a contact lens, even with 0 Dpt. refractive power, can alter the systems optical properties. In this investigation we examined the effective focal length change and the aberrations that arise from use of a contact lens. First, focal length changes were simulated with a Zemax mouse eye model. Then ocular aberrations with and without a 0 Dpt. contact lens were measured with a Shack‐Hartmann wavefront sensor (SHWS) in a customized AO‐SLO system. Total RMS wavefront errors were measured for two groups of mice (14‐month, and 2.5‐month‐old), decomposed into 66 Zernike aberration terms, and compared. These data revealed that vertical coma and spherical aberrations were increased with use of a contact lens in our system. Based on the ocular wavefront data we evaluated the effect of the contact lens on the imaging system performance as a function of the pupil size. Both RMS error and Strehl ratios were quantified for the two groups of mice, with and without contact lenses, and for different input beam sizes. These results provide information for determining optimum pupil size for retinal imaging without adaptive optics, and raise critical issues for design of mouse optical imaging systems that incorporate contact lenses. HIGHLIGHTSContact lens induced changes to mouse ocular aberrations are studied in detail using simulation and experiments.Application of the contact lens increases the effective focal length of the mouse eye and affects system resolution.Contact lens introduce two major aberrations: spherical and vertical coma (if not aligned properly).High spatial and temporal wavefront sensing allows accurate mapping of mouse eye aberrations, enabling photoreceptor imaging.