The adult zebrafish retina: In vivo optical sectioning with Confocal Scanning Laser Ophthalmoscopy and Spectral-Domain Optical Coherence Tomography.
ABSTRACT: Non-invasive imaging is an invaluable diagnostic tool in ophthalmology. Two imaging devices, the scanning laser ophthalmoscope (SLO) and spectral domain optical coherence tomography (SDOCT), emerged from the clinical realm to provide research scientists with a real-time view of ocular morphology in living animals. We utilized these two independent imaging modalities in a complementary manner to perform in vivo optical sectioning of the adult zebrafish retina. Due to the very high optical power of the zebrafish lens, the confocal depth of field is narrow, allowing for detailed en face views of specific retinal layers, including the cone mosaic. Moreover, we demonstrate that both native reflectance, as well as fluorescent features observed by SLO, can be combined with axial in-depth information obtained by SDOCT. These imaging approaches can be used to screen for ocular phenotypes and monitor retinal pathology in a non-invasive manner.
Project description:Scanning laser ophthalmoscopy (SLO) and spectral domain optical coherence tomography (SDOCT) have become essential clinical diagnostic tools in ophthalmology by allowing for video-rate noninvasive en face and depth-resolved visualization of retinal structure. Current generation multimodal imaging systems that combine both SLO and OCT as a means of image tracking remain complex in their hardware implementations. Here, we combine a spectrally encoded confocal scanning laser ophthalmoscope (SECSLO) with an ophthalmic SDOCT system. This novel implementation of an interlaced SECSLO-SDOCT system allows for video-rate SLO fundus images to be acquired alternately with high-resolution SDOCT B-scans as a means of image aiming, guidance, and registration as well as motion tracking. The system shares the illumination source, detection system, and scanning optics between both SLO and OCT as a method of providing a simple multimodal ophthalmic imaging system that can readily be implemented as a table-top or hand-held device.
Project description: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.
Project description:Photoacoustic ophthalmoscopy (PAOM) is a novel imaging technology that measures optical absorption in the retina. The capability of PAOM can be further enhanced if it could image mouse eyes, because mouse models are widely used for various retinal diseases. The challenges in achieving high-quality imaging of mouse retina, however, come from the much smaller eyeball size. Here, we report an optimized imaging system, which integrates PAOM, spectral-domain optical coherence tomography (SD-OCT), and autofluorescence-scanning laser ophthalmoscopy (AF-SLO), for mouse eyes. Its multimodal capability was demonstrated by imaging transgenic Nrl-GFP mice that express green fluorescent protein (GFP) in photoreceptors. SD-OCT provided guidance of optical alignment for PAOM and AF-SLO, and complementary contrast with high depth-resolution retinal cross sections. PAOM visualized the retinal vasculature and retinal pigment epithelium melanin, and AF-SLO measured GFP-expressing in retinal photoreceptors. The in vivo imaging results were verified by histology and confocal microscopy.
Project description:Over the past 3 decades the zebrafish (Danio rerio) has become an important biomedical research species. As their use continues to grow additional techniques and tools will be required to keep pace with ongoing research using this species. In this paper we describe a novel method for in vivo imaging of the retinal vasculature in adult animals using a commercially available confocal scanning laser ophthalmoscope (SLO). With this instrumentation, we demonstrate the ability to distinguish diverse vascular phenotypes in different transgenic GFP lines. In addition this technology allows repeated visualization of the vasculature in individual zebrafish over time to document vascular leakage progression and recovery induced by intraocular delivery of proteins that induce vascular permeability. SLO of the retinal vasculature was found to be highly informative, providing images of high contrast and resolution that were capable of resolving individual vascular endothelial cells. Finally, the procedures required to acquire SLO images from zebrafish are non-invasive, simple to perform and can be achieved with low animal mortality, allowing repeated imaging of individual fish.
Project description:<h4>Purpose</h4>To conduct high-resolution imaging of the retinal nerve fiber layer (RNFL) in normal eyes using adaptive optics scanning laser ophthalmoscopy (AO-SLO).<h4>Methods</h4>AO-SLO images were obtained in 20 normal eyes at multiple locations in the posterior polar area and a circular path with a 3-4-mm diameter around the optic disc. For each eye, images focused on the RNFL were recorded and a montage of AO-SLO images was created.<h4>Results</h4>AO-SLO images for all eyes showed many hyperreflective bundles in the RNFL. Hyperreflective bundles above or below the fovea were seen in an arch from the temporal periphery on either side of a horizontal dividing line to the optic disc. The dark lines among the hyperreflective bundles were narrower around the optic disc compared with those in the temporal raphe. The hyperreflective bundles corresponded with the direction of the striations on SLO red-free images. The resolution and contrast of the bundles were much higher in AO-SLO images than in red-free fundus photography or SLO red-free images. The mean hyperreflective bundle width around the optic disc had a double-humped shape; the bundles at the temporal and nasal sides of the optic disc were narrower than those above and below the optic disc (P<0.001). RNFL thickness obtained by optical coherence tomography correlated with the hyperreflective bundle widths on AO-SLO (P<0.001)<h4>Conclusions</h4>AO-SLO revealed hyperreflective bundles and dark lines in the RNFL, believed to be retinal nerve fiber bundles and Müller cell septa. The widths of the nerve fiber bundles appear to be proportional to the RNFL thickness at equivalent distances from the optic disc.
Project description:To assess the combined diagnostic power of frequency-doubling technique (FDT)-perimetry and retinal nerve fibre layer (RNFL) thickness measurements with spectral domain optical coherence tomography (SDOCT).The study included 330 experienced participants in five age-related groups: 77 'preperimetric' open-angle glaucoma (OAG) patients, 52 'early' OAG, 50 'moderate' OAG, 54 ocular hypertensive patients, and 97 healthy subjects. For glaucoma assessment in all subjects conventional perimetry, evaluation of fundus photographs, FDT-perimetry and RNFL thickness measurement with SDOCT was done. Glaucomatous visual field defects were classified using the Glaucoma Staging System. FDT evaluation used a published method with casewise calculation of an 'FDT-score', including all missed localized probability levels. SDOCT evaluation used mean RNFL thickness and a new individual SDOCT-score considering normal confidence limits in 32 sectors of a peripapillary circular scan. To examine the joined value of both methods a combined score was introduced. Significance of the difference between Receiver-operating-characteristic (ROC) curves was calculated for a specificity of 96%.Sensitivity in the preperimetric glaucoma group was 44% for SDOCT-score, 25% for FDT-score, and 44% for combined score, in the early glaucoma group 83, 81, and 89%, respectively, and in the moderate glaucoma group 94, 94, and 98%, respectively, all at a specificity of 96%. ROC performance of the newly developed combined score is significantly above single ROC curves of FDT-score in preperimetric and early OAG and above RNFL thickness in moderate OAG.Combination of function and morphology by using the FDT-score and the SDOCT-score performs equal or even better than each single method alone.
Project description:PURPOSE:To seek pathways of retinal pigment epithelium (RPE) fate in age-related macular degeneration via a morphology grading system; provide nomenclature, visualization targets, and metrics for clinical imaging and model systems. METHODS:Donor eyes with geographic atrophy (GA) or choroidal neovascularization (CNV) and one GA eye with previous clinical spectral-domain optical coherence tomography (SDOCT) imaging were processed for histology, photodocumented, and annotated at predefined locations. Retinal pigment epithelial cells contained spindle-shaped melanosomes, apposed a basal lamina or basal laminar deposit (BLamD), and exhibited recognizable morphologies. Thicknesses and unbiased estimates of frequencies were obtained. RESULTS:In 13 GA eyes (449 locations), 'Shedding,' 'Sloughed,' and 'Dissociated' morphologies were abundant; 22.2% of atrophic locations had 'Dissociated' RPE. In 39 CNV eyes (1363 locations), 37.3% of locations with fibrovascular/fibrocellular scar had 'Entombed' RPE; 'Sloughed,' 'Dissociated,' and 'Bilaminar' morphologies were abundant. Of abnormal RPE, CNV and GA both had ~35% 'Sloughed'/'Intraretinal,' with more Intraretinal in CNV (9.5% vs. 1.8%). 'Shedding' cells associated with granule aggregations in BLamD. The RPE layer did not thin, and BLamD remained thick, with progression. Granule-containing material consistent with three morphologies correlated to SDOCT hyperreflective foci in the previously examined GA patient. CONCLUSIONS:Retinal pigment epithelium morphology indicates multiple pathways in GA and CNV. Atrophic/scarred areas have numerous cells capable of transcribing genes and generating imaging signals. Shed granule aggregates, possibly apoptotic, are visible in SDOCT, as are 'Dissociated' and 'Sloughed' cells. The significance of RPE phenotypes is addressable in longitudinal, high-resolution imaging in clinic populations. Data can motivate future molecular phenotyping studies.
Project description: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.
Project description:Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.
Project description:Purpose:To characterize hallmark diabetic retinopathy (DR) lesions utilizing adaptive optics scanning laser ophthalmoscopy (AOSLO) and to compare AOSLO findings with those on standard imaging techniques. Methods:Cross-sectional study including 35 eyes of 34 study participants. AOSLO confocal and multiply scattered light (MSL) imaging were performed in eyes with DR. Color fundus photographs (CF), infrared images of the macula (Spectralis, Heidelberg), and Spectralis spectral domain optical coherence tomography SDOCT B-scans of each lesion were obtained and registered to corresponding AOSLO images. Main Outcome Measures:Individual lesion characterization by AOSLO imaging. AOSLO appearance was compared with CF and SDOCT imaging. Results:Characterized lesions encompassed 52 microaneurysms (MA), 20 intraretinal microvascular abnormalities (IRMA), 7 neovascularization (NV), 11 hard exudates (HE), 5 dot/blot hemorrhages (HEM), 4 cotton wool spots (CWS), and 14 intraretinal cysts. AOSLO allowed assessment of perfusion in vascular lesions and enabled the identification of vascular lesions that could not be visualized on CF or SDOCT. Conclusions:AOSLO imaging provides detailed, noninvasive in vivo visualization of DR lesions enhancing the assessment of morphological characteristics. These unique AOSLO attributes may enable new insights into the pathological changes of DR in response to disease onset, development, regression, and response to therapy.