Mayank Goswami

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.

Protective Effect of Intravitreal Administration of Exosomes Derived from Mesenchymal Stem Cells on Retinal Ischemia

ABSTRACT Purpose: Exosomes derived from human mesenchymal stem cells (hMSCs) cultured under hypoxic conditions contain proteins and growth factors that promote angiogenesis. This study investigated the effect of intravitreal administration of these exosomes on retinal ischemia using a murine model. Methods: Oxygen-induced retinopathy (OIR) was induced by exposing one-week-old male C57BL/6J mice to 5 days of 75% hyperoxic conditioning, and returning to room air. After hyperoxic conditioning, the right eye of each mouse was injected intravitreally with 1 µl saline or exosomes derived from hMSCs and compared to control mice of the same age raised in room air without OIR injected intravitreally with saline. Two weeks post-injection, fluorescein angiography (FA) and phase-variance optical coherence tomography angiography (pvOCTA) were used to assess retinal perfusion. Retinal thickness was determined by OCT. The extent of retinal neovascularization was quantitated histologically by counting vascular nuclei on the retinal surface. Results: Among eyes with OIR, intravitreal exosome treatment partially preserved retinal vascular flow in vivo and reduced associated retinal thinning; retinal thickness on OCT was 111.1 ± 7.4µm with saline versus 132.1 ± 11.6µm with exosome, p < 0.001. Retinal neovascularization among OIR eyes was reduced with exosome treatment when compared to saline-treated eyes (7.75 ± 3.68 versus 2.68 ± 1.35 neovascular nuclei per section, p < 0.0001). No immunogenicity or ocular/systemic adverse effect was associated with intravitreal exosome treatment. Conclusions: Intravitreal administration of exosomes derived from hMSCs was well tolerated without immunosuppression and decreased the severity of retinal ischemia in this murine model. This appealing novel non-cellular therapeutic approach warrants further exploration.

A New Grid-Based Tomographic Method for Two-Phase Flow Measurements

Abstract Iterative algorithms for computerized tomography reconstruction employ a variety of grids, interpolation techniques, and solution procedures. A new projection-intersection (PI) grid is presented in this work. It comprises all the intersection points between the projection rays passing through the object. A few advantages include (a) a user-independent discretization process and (b) a reduction in reconstruction error caused by nonparticipating nodes. Computerized tomography reconstruction results by PI are compared with existing conventional grids. The multiplicative algebraic reconstruction technique (MART) and entropy maximization are used as solution techniques. We note that for simulated data, the PI grid gives better results when compared with the square-pixel grid. Two different sets of experimental data (obtained previously for a mercury-nitrogen flow loop and one with a known specimen with a static known profile) are processed with the above-mentioned options. A basic theoretical model (but experimentally correlated) is also used to verify the void reference level. Computerized tomography results for experimental projection data indicate a trend similar to the previous MART results, but a major difference is visible in the void-fraction distributions. This fact is important, as heat transfer coefficients are strongly dependent on the distribution of voids.

The Photosensitivity of Rhodopsin Bleaching and Light-Induced Increases of Fundus Reflectance in Mice Measured In Vivo With Scanning Laser Ophthalmoscopy

Purpose To quantify bleaching-induced changes in fundus reflectance in the mouse retina. Methods Light reflected from the fundus of albino (Balb/c) and pigmented (C57Bl/6J) mice was measured with a multichannel scanning laser ophthalmoscopy optical coherence tomography (SLO-OCT) optical system. Serial scanning of small retinal regions was used for bleaching rhodopsin and measuring reflectance changes. Results Serial scanning generated a saturating reflectance increase centered at 501 nm with a photosensitivity of 1.4 × 10−8 per molecule μm2 in both strains, 2-fold higher than expected were irradiance at the rod outer segment base equal to that at the retinal surface. The action spectrum of the reflectance increase corresponds to the absorption spectrum of mouse rhodopsin in situ. Spectra obtained before and after bleaching were fitted with a model of fundus reflectance, quantifying contributions from loss of rhodopsin absorption with bleaching, absorption by oxygenated hemoglobin (HbO2) in the choroid (Balb/c), and absorption by melanin (C57Bl/6J). Both mouse strains exhibited light-induced broadband reflectance changes explained as bleaching-induced reflectivity increases at photoreceptor inner segment/outer segment (IS/OS) junctions and OS tips. Conclusions The elevated photosensitivity of rhodopsin bleaching in vivo is explained by waveguide condensing of light in propagation from rod inner segment (RIS) to rod outer segment (ROS). The similar photosensitivity of rhodopsin in the two strains reveals that little light backscattered from the sclera can enter the ROS. The bleaching-induced increases in reflectance at the IS/OS junctions and OS tips resemble results previously reported in human cones, but are ascribed to rods due to their 30/1 predominance over cones in mice and to the relatively minor amount of cone M-opsin in the regions scanned.

Nonuniform Arrangement of Emitter-Receiver Pairs Arrangement and Compact Ultrasonic Tomography Setup

Arrangements of emitter-receiver pairs with two possibilities: nonuniform and uniform locations, are studied. Ultrasonic computerized tomographic (UCT) setups are developed with these configurations for parallel and fan beam scanning modes. Optimization is used to determine the best nonuniform arrangement. The CT reconstructions with nonuniform emitter-receiver pair arrangements from the data acquired via computer generated pixel images and real world specimens show promising results compared with their uniform arrangement. A flow channel, e.g., a metal pipe with small ratio of pipe diameter to emitter/receiver aperture, imposes restriction on the number of emitter-receiver pairs. A practical application, for void-fraction estimation for water-air flow, is investigated with the best design. Reconstructed flow profiles and estimated air fractions match with 3-D numerical simulation obtained using FLUENT 14. Cost of a focused acoustic emitter/receiver pair and its noise response depends on its focal length. The optimized nonuniform design can be best suited for emitter/receiver pairs of small focal lengths and can aid in developing low budget and compact UCT scanners.

Optimal Spatial Filtering Schemes and Compact Tomography Setups

ABSTRACT Three compact computerized tomography (CT) scanner prototypes are established and tested for acceptable reconstruction results. Performance of conventional iterative reconstruction algorithm is enhanced via incorporating a spatial filtering/masking step. Generally, these masking strategies incorporate an arbitrary (3 3 or 2 2) size of square averaging mask to subdue the ill-posedness. Three different spatial filtering schemes are tested in this work. The objective is to remove any dependency on a user for deciding an appropriate masking parameter. The outcome of the simulation study is successfully verified for three real data situations using three specimens with pre-assigned/known inner profile. Such austere scanning situations arise in real-time environment especially for undetachable/fixed small size objects situated in inaccessible locations. The present study encourages the development of low budget CT setups.

Fluorescent scanning laser ophthalmoscopy for cellular resolution in vivo mouse retinal imaging: benefits and drawbacks of implementing adaptive optics

Scanning Laser Ophthalmoscopy (SLO) is a very important imaging tool in ophthalmology research. By combing with Adaptive Optics (AO) technique, AO-SLO can correct for ocular aberrations resulting in cellular level resolution, allowing longitudinal studies of single cells morphology in the living eyes. The numerical aperture (NA) sets the optical resolution that can be achieve in the “classical” imaging systems. Mouse eye has more than twice NA of the human eye, thus offering theoretically higher resolution. However, in most SLO based imaging systems the imaging beam size at mouse pupil sets the NA of that instrument, while most of the AO-SLO systems use almost the full NA of the mouse eye. In this report, we first simulated the theoretical resolution that can be achieved in vivo for different imaging beam sizes (different NA), assumingtwo cases: no aberrations and aberrations based on published mouse ocular wavefront data. Then we imaged mouse retinas with our custom build SLO system using different beam sizes to compare these results with theory. Further experiments include comparison of the SLO and AO-SLO systems for imaging different type of fluorescently labeled cells (microglia, ganglion, photoreceptors, etc.). By comparing those results and taking into account systems complexity and ease of use, the benefits and drawbacks of two imaging systems will be discussed.

Methods for non-surgical cancer nano-theranostics of ocular tumors in the mouse eye (Conference Presentation)

We will present our results of evaluating the feasibility of using the mouse eye as a window for non-invasive, long-term, optical investigation of xenograft models, using multimodal, cellular-resolution ocular imaging. As an “approachable part of the brain”, the retina allows examination of such issues as drug delivery across the blood retinal barrier (BRB) and blood brain barrier (BBB). Our custom-built wide-field SLO/OCT provided repeatable in vivo imaging over many weeks, allowing quantitative tracking of tumor growth, the delivery of theranostic nanoparticles, and the measurement of tumor microenvironment responses. Additionally, we were able to specifically control the spatial extent of light activated photodynamic therapy (PDT) and photothermal therapy (PTT) via efficient free radical and heat generation at the tumor site, respectively.

Light induced increases of photoreceptor layer reflectance in response to rhodopsin bleaching in mice measured in vivo with optical coherence tomography (Conference Presentation)

We have recently reported observations of light-induced broadband fundus reflectance changes in two most commonly used strains of laboratory mice, C57Bl/6J (pigmented) and Balb/c (un- pigmented albino). The action spectrum of the reflectance increase corresponded to the absorption spectrum of mouse rhodopsin in situ. Spectral changes in mouse fundus reflectivity were calculated from measurements made by broadband spectrometer, interfaced with our mouse retinal SLO system, obtained before and after bleaching. This results were fitted with a model of mouse fundus reflectance, quantifying contributions from loss of rhodopsin absorption with bleaching, absorption by oxygenated hemoglobin (HbO2) in the choroid (Balb/c), and absorption by melanin (C57Bl/6J) additionally both mouse strains exhibited light-induced broadband reflectance changes explained as bleaching-induced reflectivity increases at photoreceptor inner segment/outer segment (IS/OS) junctions and OS tips. Here we present results investigating the kinetics of the increases in reflectivity with Optical Coherence Tomography operating in a 780-950 nm band.

Visualization of chorioretinal vasculature in mice in vivo using a combined OCT/SLO imaging system

Chorioretinal blood vessel morphology in mice is of great interest to researchers studying eye disease mechanisms in animal models. Two leading retinal imaging modalities -- Optical Coherence Tomography (OCT) and Scanning Laser Ophthalmoscopy (SLO) -- have offered much insight into vascular morphology and blood flow. OCT “flow-contrast” methods have provided detailed mapping of vascular morphology with micrometer depth resolution, while OCT Doppler methods have enabled the measurement of local flow velocities. SLO remains indispensable in studying blood leakage, microaneurysms, and the clearance time of contrast agents of different sizes. In this manuscript we present results obtained with a custom OCT/SLO system applied to visualize the chorioretinal vascular morphology of pigmented C57Bl/6J and albino nude (Nu/Nu) mice. Blood perfusion maps of choroidal vessels and choricapillaris created by OCT and SLO are presented, along with detailed evaluation of different OCT imaging parameters, including the use of the scattering contrast agent Intralipid. Future applications are discussed.