Adaptive optics scanning laser ophthalmoscopy and optical coherence tomography (AO-SLO-OCT) system for in vivo mouse retina imaging.

Optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) are imaging technologies invented in the 1980s that have revolutionized the field of in vivo retinal diagnostics and are now commonly used in ophthalmology clinics as well as in vision science research. Adaptive optics (AO) technology enables high-fidelity correction of ocular aberrations, resulting in improved resolution and sensitivity for both SLO and OCT systems. The potential of gathering multi-modal cellular-resolution information in a single instrument is of great interest to the ophthalmic imaging community. Although similar instruments have been developed for imaging the human retina, developing such a system for mice will benefit basic science research and should help with further dissemination of AO technology. Here, we present our work integrating OCT into an existing mouse retinal AO-SLO system, resulting in a multi-modal AO-enhanced imaging system of the living mouse eye. The new system allows either independent or simultaneous data acquisition of AO-SLO and AO-OCT, depending on the requirements of specific scientific experiments. The system allows a data acquisition speed of 200 kHz A-scans/pixel rate for OCT and SLO, respectively. It offers ∼6 µm axial resolution for AO-OCT and a ∼1 µm lateral resolution for AO-SLO-OCT imaging.

Pseudo-real-time retinal layer segmentation for high-resolution adaptive optics optical coherence tomography.

We present a pseudo-real-time retinal layer segmentation for high-resolution Sensorless Adaptive Optics-Optical Coherence Tomography (SAO-OCT). Our pseudo-real-time segmentation method is based on Dijkstra's algorithm that uses the intensity of pixels and the vertical gradient of the image to find the minimum cost in a geometric graph formulation within a limited search region. It segments six retinal layer boundaries in an iterative process according to their order of prominence. The segmentation time is strongly correlated to the number of retinal layers to be segmented. Our program permits en face images to be extracted during data acquisition to guide the depth specific focus control and depth dependent aberration correction for high-resolution SAO-OCT systems. The average processing times for our entire pipeline for segmenting six layers in a retinal B-scan of 496 × 400 and 240 × 400 pixels are around 25.60 and 13.76 ms, respectively. When reducing the number of layers segmented to only two layers, the time required for a 240 × 400 pixel image is 8.26 ms.

Adaptive optics in the mouse eye: wavefront sensing based vs. image-guided aberration correction.

Adaptive Optics (AO) is required to achieve diffraction limited resolution in many real-life imaging applications in biology and medicine. AO is essential to guarantee high fidelity visualization of cellular structures for retinal imaging by correcting ocular aberrations. Aberration correction for mouse retinal imaging by direct wavefront measurement has been demonstrated with great success. However, for mouse eyes, the performance of the wavefront sensor (WFS) based AO can be limited by several factors including non-common path errors, wavefront reconstruction errors, and an ill-defined reference plane. Image-based AO can avoid these issues at the cost of algorithmic execution time. Furthermore, image-based approaches can provide improvements to compactness, accessibility, and even the performance of AO systems. Here, we demonstrate the ability of image-based AO to provide comparable aberration correction and image resolution to the conventional Shack-Hartmann WFS-based AO approach. The residual wavefront error of the mouse eye was monitored during a wavefront sensorless optimization to allow comparison with classical AO. This also allowed us to improve the performance of our AO system for small animal retinal imaging.

Non-invasive cellular-resolution retinal imaging with two-photon excited fluorescence.

Two-photon excited fluorescence (TPEF) imaging of the retina is a developing technique that provides non-invasive compound-specific measurements from the retina. In this report, we demonstrate high-resolution TPEF imaging of the mouse retina using sensorless adaptive optics (SAO) and optical coherence tomography (OCT). A single near-infrared light source was used for simultaneous multi-modal imaging with OCT and TPEF. The image-based SAO could be performed using the en face OCT or the TPEF for aberration correction. Our results demonstrate OCT and TPEF for angiography. Also, we demonstrate non-invasive cellular-resolution imaging of fluorescently labelled cells and the Retinal Pigment Epithelium (RPE) mosaic.

Multi-scale and -contrast sensorless adaptive optics optical coherence tomography.

BACKGROUND: The roles of the retinal microvasculature and the retinal pigment epithelium (RPE) in maintaining the health and metabolic activity of the retina lend great clinical value to their high-resolution visualization. METHODS: By integrating polarization diversity detection (PDD) into multi-scale and -contrast sensorless adaptive optics optical coherence tomography (MSC-SAO-OCT), we have developed a novel multi-contrast SAO OCT system for imaging pigment in the RPE as well as flow in the retinal capillaries using OCT angiography (OCTA). Aberration correction was performed based on the image quality using transmissive deformable optical elements. RESULTS: MSC-SAO-OCTA imaging was performed at multiple fields-of-view (FOVs) with adjustable numerical aperture (NA). Retinal flow and RPE structural images for in vivo healthy and pathological human posterior eyes were demonstrated to show clinical feasibility of the system. CONCLUSIONS: High-resolution imaging of retinal vasculature at both large and small FOVs, as well as characterization of RPE topology and deformation, enables more sophisticated and concise investigation of retinal pathologies for in vivo human imaging. MSC imaging may permit detection and analysis of even subtle deformations in the RPE layer using a single instrument.

Sensorless adaptive optics multimodal en-face small animal retinal imaging.

Vision researchers often use small animals due to the availability of many transgenic strains that model human diseases or express biomarkers. Adaptive optics (AO) enables non-invasive single-cell imaging in a living animal but often results in high system complexity. Sensorless AO (SAO) can provide depth-resolved aberration correction with low system complexity. We present a multi-modal sensorless AO en face retina imaging system that includes optical coherence tomography (OCT), OCT-angiography, confocal scanning laser ophthalmoscopy (SLO), and fluorescence detection. We present a compact lens-based imaging system design that allows for a 50-degree maximum field of view (FOV), which can be reduced to the region of interest to perform SAO with the modality of choice. The system performance was demonstrated on wild type mice (C57BL/6J), and transgenic mice with GFP labeled cells. SAO SLO was used for imaging microglia (Cx3cr1-GFP) over ~1 hour, where dynamics of the microglia branches were clearly observed. Our results also include volumetric cellular imaging of microglia throughout the inner retina.

Ensuring Patient Safety in Emergency Peripheral Ultrasound-Guided Nerve Blocks: An Evaluation of a Quality Improvement/Patient Safety Initiative.

CONTEXT: During the past two decades, bedside ultrasound has revolutionized the practice of emergency medicine. Physicians are now expected to be competent in utilizing ultrasound skills, for patients presenting with conditions ranging from trauma to skin evaluations. The overall purpose of this quality improvement/patient safety (QIPS) project was to evaluate the effectiveness of a pair of five-hour, hands-on didactic/training sessions, aimed at preparing a sample of emergency medicine physicians, residents and medical students to perform peripheral ultrasound-guided nerve blocks. METHODS: The study location was set in a community-based emergency medicine program in Pontiac, Michigan. Data was collected from N = 54 emergency medicine residents, physicians and medical students. Data was collected from two training sessions in November 2017 and January 2018. The training consisted of a 12-question pre-test, followed by five hours of hands on & didactic training, with a subsequent post-test containing the same questions. RESULTS: The authors compiled the data from both training sessions and found that the participants had an average correct percentage of 5.52 of 12 (46%) on the pre-test. After attending the training session, participants had an overall correct percentage of 9.24 of 12 (77%) on the post-test. This pre-to post-training increase of the mean scores was statistically significant, t (53) = -10.76 (p < 0.01), with an effect size (Cohen's d) of 1.82. Post hoc power calculations utilizing the d = 1.82 effect size revealed statistical power (1- ß) of 100%. CONCLUSIONS: The results of this QIPS evaluation project suggest that emergency physicians, residents and medical students may achieve an improved understanding of key ultrasound-guided nerve block material after a single five-hour session of hands-on training and didactics. Going forward, additional studies employing larger sample sizes that allow for outcome stratification by group (emergency physicians, residents, or medical students) along with relevant demographic variables (age, years in practice, etc.) in similar settings are needed to further verify these findings.

Visible light sensorless adaptive optics for retinal structure and fluorescence imaging.

Optical coherence tomography (OCT) has emerged as a powerful imaging instrument and technology in biomedicine. OCT imaging is predominantly performed using wavelengths in the near infrared; however, visible light (VIS) has been recently employed in OCT systems with encouraging results for high-resolution retinal imaging. Using a broadband supercontinuum VIS source, we present a sensorless adaptive optics (SAO) multimodal imaging system driven by VIS-OCT for volumetric retinal structural imaging, followed by the acquisition of fluorescence emission. The coherence-gated, depth-resolved VIS-OCT images used for image-guided SAO aberration correction enable high-resolution structural and fluorescence imaging.

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

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 system's 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.

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.

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 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.