This study is supported by the Glaucoma Research Foundation Shaffer Grant, 4-CA Cavalier Collaborative Award, R01EY029121, R01EY035088, and Knights Templar Eye Foundation.&#8195;

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Virginia and conformed to the guideline on Use of Animals from the National Institute of Health (NIH). See the Table of Materials for details related to all materials, reagents, and instruments used in this protocol.
1. In vivo vis-OCT imaging
<ol>
	<li>The vis-OCT system
	<ol>
		<li>Image the mice eyes using a small animal vis-OCT system that uses a supercontinuum light source, which delivers visible-light illumination between 480 nm and 650 nm. Ensure that the power incident on the cornea is 1 mW and use an A-line rate of 25 kHz and an integration time of 39.3 &#181;s per A-line.</li>
		<li>Ensure that the spectral detection range of the spectrometer is between 508 nm and 613 nm, which provides an axial resolution of 1.3 &#181;m in the retina. The total imaging volume is approximately 700 &#181;m (x) x 700 &#181;m (y) x 1,500 &#181;m (z). The lateral resolution is between 4.5 &#181;m at the center of the field of view and 8.7 &#181;m at 350 &#181;m from the center11,13.</li>
	</ol>
	</li>
	<li>Mouse anesthesia
	<ol>
		<li>Anesthetize mice of C57BL/6 background and either sex with an intraperitoneal injection of Ketamine (114 mg/kg) and Xylazine (17 mg/kg) cocktail and dilate their pupils using 1% tropicamide drops. Confirm adequate anesthetization by loss of pedal reflex after a firm toe pinch.</li>
		<li>During imaging, keep the mouse warm by using an infrared heat lamp. After each image acquisition, apply artificial tears to prevent corneal dehydration.</li>
	</ol>
	</li>
	<li>Positioning of the mouse for imaging
	<ol>
		<li>Place the anesthetized mouse on the animal holder and keep the mouse in position with the help of two Velcro straps. 
		NOTE: The animal holder allows movement in three dimensions (vertical adjustment, fine horizontal adjustment, as well as pitch and yaw adjustments) to place the laser into the mouse&#39;s eye.</li>
	</ol>
	</li>
	<li>Adjusting imaging parameters
	<ol>
		<li>Turn on the computer and open the referenced software, which will turn on the laser automatically.</li>
		<li>Adjust the animal holder until the laser is stable and centered into the mouse&#39;s eye. Visualize the posterior part of the eye through an En Face preview in the software interface, the field of view (FOV) of the superficial vascular plexus, and a B-scan, the cross-section of the retina within the FOV.</li>
		<li>Acquire a vis-OCT volume by clicking on the Acquire button on the software interface after performing minor adjustments of the optical focus, which consists of 512 A-lines/B-scan and 512 B-scans/volume. 
		NOTE: This process takes ~10.5 s. Image acquisition is guided by a built-in quality index (QI) estimator to ensure that images below a predetermined threshold (QI &#60; 45) are not included.
		<ol>
			<li>For each mouse, acquire four vis-OCT volumes from the same eye. Align the optic nerve head (ONH) in each of the four corners in the FOV to cover different areas of the retina. 
			NOTE: Such placement minimizes the retinal curvature, which maximizes RNFL reflectance throughout the FOV. It requires ~1 min to reposition the eye between each acquisition (Figure 1A,B).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Vis-OCTF analysis 
	NOTE: MATLAB is used to perform image analysis.
	<ol>
		<li>To generate vis-OCT fibergrams from the vis-OCT volume, use an intensity-based threshold method (MATLAB code line&#160;808) to detect the surface of the retina. 
		NOTE: These lines use the imadjust function to adjust the intensity values of the bscan. The [0.0087 0.08] argument passed to imadjust specifies the range of intensities to map to the full dynamic range of the output image.</li>
		<li>Crop the RNFL by selecting the first ~16 &#181;m in depth. See Matlab code line 782. 
		NOTE: The typical in vivo RNFL thickness in an adult wild-type C57BL/6 mouse is ~14 &#181;m and can vary among different segmentation methods13.</li>
		<li>Change the paths of the RAW file (line 9), Files for reconstruction (line 11), and file name (line 15), click Run, and wait for the OCT image to be analyzed by the MATLAB code. Calculate the mean intensity projection along the axial (z) direction (MATLAB code lines 905-908) to generate the fibergram image composed of RGC axon bundles and surrounding vasculature. Montage the four images after fibergram processing for each FOV by aligning the blood vessels with a graphics editor of choice, covering ~1.2 x 1.2 mm in total. The RAW files are usually saved in the folder: Halo Data, under the date of OCT imaging (e.g., 0606 Opticent). 
		&#8203;NOTE: The MATLAB codes are available in Supplementary File 1.</li>
	</ol>
	</li>
</ol>
2. Ex vivo confocal imaging
<ol>
	<li>Mouse euthanasia
	<ol>
		<li>After acquiring vis-OCT data, euthanize the mice with a mixture of pentobarbital sodium (390 mg/mL) and phenytoin sodium (50mg/mL).&#160;Phenytoin sodium functions by amplifying the effects of pentobarbital sodium, which has been widely applied in rodent euthanasia14,15. For each mouse, use 0.2 mL/20 g of the diluted mixture (156 mg/mL) and perfuse with 20 mL of phosphate-buffered saline (PBS) and then, 20 mL of 4% paraformaldehyde (PFA) in PBS16,17.</li>
	</ol>
	</li>
	<li>Eye dissection and orientation
	<ol>
		<li>Enucleate the eyes and make a mark on the temporal side to indicate the orientation.</li>
		<li>After carefully removing the anterior chamber,&#160;the ocular lens, vitreous, and fix the eyecups in PFA for 30 min.</li>
		<li>Wash the eyecups with PBS for 30 min, and replace the PBS solution 3x during the washing. Then, wash with 0.5% Triton X-100 in PBS (pH 7.5) for 30 min.</li>
		<li>Incubate the eyecups in blocking buffer (5% donkey serum with 2.5% bovine serum albumin and 0.5% Triton X-100 in Tris-buffered saline [pH 7.5]) for 2 h at room temperature.</li>
	</ol>
	</li>
	<li>Immunostaining 
	NOTE: The eyecups are now ready to be stained with the primary antibodies of mouse anti-Tuj1 to detect RGC axon bundles and rat anti-ICAM-2 to detect blood vessels.
	<ol>
		<li>Incubate the eye cups overnight with the primary antibodies, mouse anti-Tuj1 (1:200 in blocking buffer) and rat anti-ICAM-2 (1:500 in blocking buffer), at 4 &#176;C.</li>
		<li>Wash the eyecups 3-5x for 1 h each time, with a phosphate-buffered saline solution containing 0.5% Triton X-100 (PBST) to minimize background and remove any unbound antibody.</li>
		<li>After washing, incubate the eye cups overnight with the secondary antibodies, donkey anti-mouse Immunoglobulin G conjugated to Alexa Fluor 488 dye (green fluorescence) and donkey anti-rat IgG conjugated to Alexa Fluor 594 dye (red fluorescence), all diluted 1:1000 in blocking buffer, at 4 &#176;C.</li>
		<li>The following day, wash the eyecups 3-5x for 1 h each with PBST to minimize background and remove unbound antibody.</li>
		<li>Transfer the eyecups to a Petri dish of PBS before the flat mount.</li>
	</ol>
	</li>
	<li>Flat-mounted retina
	<ol>
		<li>After the immunostaining process, isolate the retinas from the eyecups under the microscope.</li>
		<li>Cut the retinas into four leaves and flat mount them with the RGC layer facing up. Leave the mark on the temporal side attached to the retina to indicate orientation.</li>
		<li>Coverslip the retinas with mounting medium13,18. Seal the slides with nail polish13,18,19.</li>
	</ol>
	</li>
	<li>Confocal imaging 
	NOTE: The image processing of confocal images was performed using ZEN Microscopy Software.
	<ol>
		<li>Turn on the confocal microscope, and under Locate mode, find the area of interest using the eyepiece of the microscope.</li>
		<li>Under acquisition mode, set up tiles to cover the whole retina and z-stack slices to cover all layers of information. Image a least 25 tiles across the whole retina to cover the total volume of 5.99 mm (x) x 5.88 mm (y) x 30 &#181;m (z) at a pixel size of 1.24 &#181;m/pixel.</li>
		<li>Project the Z-stack slices to create two-dimensional en face confocal microscopy images (Figure 1C,D)11,13,19,20,21.</li>
	</ol>
	</li>
</ol>
3. Alignment of in vivo and ex vivo images
<ol>
	<li>After processing the fibergram, create a composite image that includes the four images acquired from each mouse by aligning all the blood vessels with a graphics editor of choice. 
	NOTE: On average, the final composite image is approximately 1.2 mm (x) x 1.2 mm (y), as shown in Figure 1B.</li>
	<li>Use the optic nerve head (ONH) and the pattern of blood vessels as landmarks to align the composite fibergram. Obtain the in vivo and ex vivo alignment by overlapping the blood vessel pattern of the composite OCT images with the confocal images of the same retina stained with ICAM-2.</li>
</ol>

Mouse Retina OCT Fibergram Alignment with Confocal Images

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible-light+optical+coherence+tomography+fibergraphy+for+quantitative+imaging+of+retinal+ganglion+cell+axon+bundles.">Visible-light optical coherence tomography fibergraphy for quantitative imaging of retinal ganglion cell axon bundles.</a> Translational Vision Science and Technology. 9 (11), (2020).</li><li>Beckmann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+the+inner+retinal+layer+structure+in+mice+after+eye-opening+using+visible-light+optical+coherence+tomography.">In vivo imaging of the inner retinal layer structure in mice after eye-opening using visible-light optical coherence tomography.</a> Experimental Eye Research. 211, 108756 (2021).</li><li>Grannonico, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+damages+in+retinal+ganglion+cell+axon+bundles+monitored+non-invasively+by+visible-light+optical+coherence+tomography+fibergraphy.">Global and regional damages in retinal ganglion cell axon bundles monitored non-invasively by visible-light optical coherence tomography fibergraphy.</a> Journal of Neuroscience. 41 (49), 10179-10193 (2021).</li><li>Allen-Worthington, K. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiopoietin-1+knockout+mice+as+a+genetic+model+of+open-angle+glaucoma.">Angiopoietin-1 knockout mice as a genetic model of open-angle glaucoma.</a> Translational Vision Science and Technology. 9 (4), (2020).</li><li>Feng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+ocular+hypertension+induces+dendritic+degeneration+of+mouse+retinal+ganglion+cells+that+depends+on+cell+type+and+location.">Sustained ocular hypertension induces dendritic degeneration of mouse retinal ganglion cells that depends on cell type and location.</a> Investigative Ophthalmology and Visual Science. 54 (2), 1106-1117 (2013).</li><li>Grannonico, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+analysis+of+retinal+ganglion+cell+damage+at+individual+axon+bundle+level+in+mice+using+visible-light+optical+coherence+tomography+fibergraphy.">Longitudinal analysis of retinal ganglion cell damage at individual axon bundle level in mice using visible-light optical coherence tomography fibergraphy.</a> Translational Vision Science and Technology. 12 (5), (2023).</li></ol>

Chang, S., None; Xu, W., None; Fan, W., None; McDaniel, J.A., None; D.A. Miller, None; M. Grannonico, None; M. Liu, None; X. Liu, None; H.F. Zhang has financial interests in Opticent Health, which did not support this work.

There are two steps in this protocol that require attention. First, it is necessary to ensure that the animal is under deep anesthesia and that their eyes are fully dilated before vis-OCT imaging. If the mice are not adequately anesthetized, their fast breathing may lead to unstable movements of the en face images, which can adversely affect the quality of the fibergram. Moreover, insufficient dilation can also have a negative impact on image quality since the iris may obstruct the light, preventing it from reac...

Retinal ganglion cells (RGCs) play a critical role in visual information processing, receiving synaptic inputs through their dendritic trees in the inner plexiform layer (IPL) and transmitting the information via their axons in the retinal nerve fiber layer (RNFL) to the brain1,2,3,4. In diseased conditions such as glaucoma, early RGC degeneration may result in subtle changes in the RNFL, the ganglion cell layer (GCL), the IPL, and the optic nerve in both patients and rodent models5,6,7,8,9. Early detection of these morphological changes in RGCs is thus essential for timely intervention to prevent RGC and vision loss.
We have recently developed a new clinical-ready imaging technology called visible-light optical coherence tomography (vis-OCT) to satisfy the need for in vivo monitoring of RGC damage. Vis-OCT improved the axial resolution, reaching 1.3 &#181;m in the retina10, 11, allowing for the visualization of individual RGC axon bundles in the RNFL. Subsequently, vis-OCT fibergraphy (vis-OCTF) was established to track and quantify RGC damage at the single axon bundle level in mice11,12,13. However, ex vivo confocal imaging of the same retina as the gold standard is often necessary to validate the in vivo findings. Therefore, this study will demonstrate how to align in vivo images acquired by vis-OCTF with ex vivo confocal images of the same mouse retina. The protocol aims to validate the in vivo findings by ex vivo confocal imaging and establish a foundation for examining the molecular and cellular changes underlying RGC damage in diseased conditions.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Halo 100</td><td>Opticent Health, Evanston, IL</td><td></td><td></td></tr><tr><td>Zeiss LSM800 microscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Drugs and antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4% paraformaldehyde (PFA)</td><td>Santz Cruz Biotechnology, SC-281692</td><td></td><td>1-2 drops</td></tr><tr><td>Bovine serum albumin powder</td><td>Fisher Scientific, BP9706-100</td><td></td><td>1:10</td></tr><tr><td>Donkey anti Mouse Alexa Fluor 488 dye</td><td>Thermo Fisher Scientific, Cat# A-21202</td><td></td><td>1:1,000</td></tr><tr><td>Donkey anti rat Alexa Fluor 594 dye</td><td>Thermo Fisher Scientific, Cat# A-21209</td><td></td><td>1:1,000</td></tr><tr><td>Euthasol (a mixture of pentobarbital sodium (390 mg/mL) and phenytoin sodium (50 mg/mL))</td><td>Covetrus, NDC 11695-4860-1</td><td></td><td>15.6 mg/mL</td></tr><tr><td>Ketamine</td><td>Covetrus, NADA043304</td><td></td><td>114 mg/kg</td></tr><tr><td>Mouse anti-Tuj1</td><td>A gift from Anthony J. Spano, University of Virginia</td><td></td><td>1:200</td></tr><tr><td>Normal donkey serum(NDS)</td><td>Millipore Sigma, S30-100 mL</td><td></td><td>1:100</td></tr><tr><td>Phosphate-buffered saline (PBS, 10x), pH 7.4&lt;br/&gt; (Contains 1370 mM NaCl, 27 mM KCl, 80 mM Na2HPO4, and 20 mM KH2PO4)</td><td>Thermo Fisher Scientific, Cat# J62036.K3</td><td></td><td>1:10</td></tr><tr><td>Rat anti-ICAM-2</td><td>BD Pharmingen, Cat#553325</td><td></td><td>1:500</td></tr><tr><td>Tropicamide drops&amp;nbsp;</td><td>Covetrus, NDC17478-102-12</td><td></td><td></td></tr><tr><td>Triton X-100&lt;br/&gt; (Reagent Grade)</td><td>VWR, CAS: 9002-93-1</td><td></td><td>1:20</td></tr><tr><td>Vectashield mounting medium</td><td>Vector Laboratories Inc. H2000-10</td><td></td><td></td></tr><tr><td>Xylazine</td><td>Covetrus, NDC59399-110-20</td><td></td><td>17 mg/kg</td></tr></tbody></table>

alignment of visible-light optical coherence tomography fibergrams with confocal images of the same mouse retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

The composite vis-OCT fibergram is compared with the corresponding confocal image of flat-mounted retina immunostained with Tuj-1 for RGC axons (Figure 1D, top panel). Axon bundles imaged by vis-OCTF can be matched with the Tu-j1-labeled axon bundles on the confocal image. Blood vessels usually exhibit distinguishable branching structures compared with surrounding axon bundles in fibergram images, which can be matched with the ICAM-2-labeled blood vessels on the confocal image (<strong class...

Watch this Scientific Journal Video about Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment at JoVE.com

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

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1.4K Views.  University of Virginia. The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images. 

Video: Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

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 challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

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

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

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

Bioengineering

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Medicine

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://appjove.unicartagenaproxy.elogim.com/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

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Chapters in this video

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

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

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

In Vivo Imaging of Cx3cr1^gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Two-Photon In Vivo Imaging of the Mouse Retina