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Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Boston University School of Medicine

Multi-modal three dimensional (3D) optical imaging combining both structural sensitivity and molecular specificity is highly desirable in biomedical research. In this paper, we present a method termed oblique scanning laser microscopy (OSLM) to combine optical coherence tomography (OCT), for simultaneously volumetric structural and molecular imaging with cellular resolution in all three dimensions. Conventional 3D laser scanning fluorescence microscopy requires repeated optical sectioning to create z-stacks in depth. Here, the use of an obliquely scanning laser eliminates the z-stacking process, then allows highly efficient 3D OCT and fluorescence imaging by using only one raster scan. The current setup provides ~3.6 × 4.2 × 6.5 μm resolution in fluorescence imaging, ~7 × 7 × 3.5 μm in OCT in three dimensions, and the current speed of imaging is up to 100 frames per second (fps) over a volume about 0.8 × 1 × 0.5 mm. We demonstrate several mechanisms for molecular imaging, including intrinsically expressed GFP fluorescence, autofluorescence from Flavin proteins, and exogenous antibody-conjugated dyes. We also demonstrate potential applications in imaging human intestinal organoids (HIOs), colon mucosa, and retina.

Oblique scanning laser microscopy for simultaneously volumetric structural and molecular imaging using only one raster scan.

In this paper, we present the first numerical study on full metrics of wavelength-dependent optical properties of melanosomes in retinal pigmented epithelial (RPE) cells. T-matrix method was used to simulate the spheroidal shapes of mature melanosomes, and the complex refractive index was calculated by a subtractive Kramers-Kronig relation for melanin. The validity of the method was first confirmed by Mie theory, and corroborated by a comparison between visible light and near infrared (NIR) optical coherence tomography (OCT) on human retinal imaging. We also studied the changes of melanosome optical properties due to melanin bleaching by numerically reducing the absorption of melanin. This study implies a unique approach to detect melanin changes specifically in RPE by a spectroscopic contrast of optical coherence tomography.

Wavelength-dependent optical properties of melanosomes in retinal pigmented epithelium and their changes with melanin bleaching: a numerical study.

While fluorescent contrast is widely used in ophthalmology, three-dimensional (3D) fluorescence retinal imaging over a large field of view (FOV) has been challenging. In this paper, we describe a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging with only one raster scan. The technique utilizes scanned oblique illumination and angled detection to obtain fluorescent cross-sectional images, analogous to optical coherence tomography (OCT) line scans (or B-scans). By breaking the coaxial optical alignment used in conventional retinal imaging modalities, depth resolution is drastically improved. To demonstrate the capability of oSLO, we have performed  volumetric fluorescein angiography (FA) of the rat retina with ~25μm depth resolution and over a 30° FOV. Using depth segmentation, oSLO can obtain high contrast images of the microvasculature down to single capillaries in 3D. The multi-modal nature of oSLO also allows for seamless combination with simultaneous OCT angiography.

Volumetric fluorescence retinal imaging  over a 30-degree field of view by oblique scanning laser ophthalmoscopy (oSLO).

Flow-mediated vasodilation (FMD) is used for assessment of vascular endothelial function in humans as a predictor of cardiovascular events. It has been challenging to carry it on preclinical murine models due to the diminutive size of the femoral artery. Here, we present a new approach to accurately measure the blood velocity and femoral artery diameters of mice by acquiring Doppler optical coherence tomography and optical coherence tomography angiography continuously within 1 single experimental scanning protocol. Using the 3-dimensional imaging and new velocity algorithm, the measurement precision of diameter, blood flow, velocity and wall shear stress are improved to 0.91%, 11.0%, 10.7% and 14.0%, respectively. FMD of healthy mouse femoral artery measured by this method was 11.96% ± 0.98%, which was blunted to 5.69% ± 0.4% by intravenous administration of endothelial nitric oxide synthase inhibitor (L-N -Nitroarginine methyl ester), in agreement with that reported in the literature.

Measurement of flow-mediated dilation of mouse femoral artery in vivo by optical coherence tomography.

Elastic light scattering spectroscopy (ELSS) has been proven a powerful method in measuring tissue structures with exquisite nanoscale sensitivity. However, ELSS contrast in the living human retina has been relatively underexplored, primarily due to the lack of imaging tools with a large spectral bandwidth. Here, we report a simple all fiber-based setup to implement dual-channel visible and near infrared (NIR) optical coherence tomography (vnOCT) for human retinal imaging, bridging over a 300nm spectral gap. Remarkably, the fiber components in our vnOCT system support single-mode propagation for both visible and NIR light, both of which maintain excellent interference efficiencies with fringe visibility of 97% and 90%, respectively. The longitudinal chromatic aberration from the eye is corrected by a custom-designed achromatizing lens. The elegant fiber-based design enables simultaneous imaging for both channels and allows comprehensive ELSS analysis on several important anatomical layers, including nerve fiber layer, outer segment of the photoreceptors and retinal pigment epithelium. This vnOCT platform and method of ELSS analysis open new opportunities in understanding structure-function relationship in the human retina and in exploring new biomarkers for retinal diseases.

Weiye Song

Department of Medicine

Boston University School of Medicine

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

Weiye Song

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medicine

Boston University School of Medicine

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

Department of Medicine