This work was supported by the Department of Veterans Affairs Rehab R&#38;D Service Career Development Awards (CDA-1, RX002111; CDA-2; RX002928) to RSA, Merit Award (RX002615) and Research Career Scientist Award (RX003134) to MTP, Career Development Award (CDA-2, RX002342) to AJF, EY028859 to MTP, NEI Core Grant P30EY006360, Research to Prevent Blindness, and Foundation Fighting Blindness.

All procedures described were approved by the Atlanta Veterans Affairs Institutional Animal Care and Use Committee and conformed to the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications, 8th edition, updated 2011).
NOTE: The SD-OCT system used to develop the protocol below is described in the Table of Materials. While some of the procedures are specific to this particular system, the overall approach can be adapted for other OCT devices and animal models. Further, in our lab, these protocols are commonly used in mice and rats; however, the overall approach can be adopted to different animal models and SD-OCT devices provided an individual has the correct lens and capabilities on their device.
1. Set up the optical coherence tomography equipment
<ol>
	<li>Open the SD-OCT software (Table of Materials).</li>
	<li>Define who is taking the OCT, the study, and the treatment arm (if relevant). Name these categories in a way that will help researchers search for the desired scans later during data analysis.
	<ol>
		<li>In the Patient/Exam tab, click Test Examiner. Select the name of the examiner. Use the Setup Examiners &#38; Physicians button to add new examiners.</li>
		<li>Click Study Name to define the study. Click the Study tab to add a new study or modify treatments in an existing study. Click to the right of Select Treatment Arm to select a treatment arm.</li>
	</ol>
	</li>
	<li>Click the Add Patient button, which is used to add a new timepoint for an entire group. When the window appears, enter ID number, First name, and Last name. Select Male or Female. Enter the Date of Birth.</li>
	<li>Click the Add Exam button to add the individual rats. To identify the rats, click on an exam. Click Edit Exam. Enter the ID number into the Enter Notes box. Click the Save Changes button.</li>
	<li>Attach the proper lens to the device (Figure 1B), select the corresponding Configuration in the software, and dial in the associated reference arm position. 
	NOTE: The SD-OCT system described has customized lenses, preset scan patterns, and reference arm settings specific to the animal species and region of eye being imaged (retina or cornea, mouse or rat). Some of these details are specific to the SD-OCT system described (see table of materials). For example, not all devices offer manual adjustment of reference arm pathlength.</li>
	<li>In the Patient/Exam tab, double click the highlighted exam to proceed to the Imaging tab and begin imaging or simply click the Imaging tab. If there is a default scan, right click to delete it.</li>
	<li>Load a pre-set Scan Protocol by clicking the Select a protocol from the list button. Alternatively, add individual scans.</li>
	<li>For rat models of glaucoma and diabetic retinopathy and mouse models of retinal degeneration, choose a pre-set that consists of four images: 2 OD and 2 OS scans. For mouse myopia, choose a pre-set that consists of 8 images: 4 OD and 4 OS scans. 
	NOTE: Pre-set imaging will be explained in more detail in Section 3. This is something each laboratory makes for themselves or with the manufacturer during on-site installation.</li>
</ol>
2. Anesthetize the animal
<ol>
	<li>Administer anesthetic.
	<ol>
		<li>Anesthetize rats with ketamine (60 mg/kg) and xylazine (7.5 mg/kg) via intraperitoneal injection.</li>
		<li>Anesthetize mice with ketamine (80 mg/kg) and xylazine (16 mg/kg) via intraperitoneal injection.</li>
		<li>Wait until animals are fully anesthetized and do not respond to toe pinch.</li>
	</ol>
	</li>
	<li>Administer pupil dilation drops (1% tropicamide). Wait for pupils to dilate before imaging. 
	NOTE: Dilation of pupils increases the field of view but is not a requirement. Local (corneal) anesthetic drops (0.5% tetracaine) to numb the eye should also be used if anything will be touching the eye (for example, if applying contact lenses or using a guide). A guide is a device that is placed over the scan head and helps beginners to line up the eye and the scan head.</li>
	<li>After anesthetizing the rodent, place the rodent in a rodent alignment system that can rotate the animal in 3-dimensional space (Figure 1A, 1C, &#38; 1D). Provide thermal support. 
	NOTE: Currently, we use rodent alignment systems for mice and rats designed and sold with the SD-OCT device.</li>
	<li>Apply liquid (e.g., saline or artificial tears) to keep the eyes lubricated. Ensure that the eye does not dry out during imaging so that the optical properties of the eye are maintained between scans (when the cornea is wet, the retina can be seen clearly).
	<ol>
		<li>Make sure to maintain moisture in the opposite eye when scanning the first eye so it does not dry out.</li>
	</ol>
	</li>
	<li>Use a delicate task wipe to wick away excess saline just before imaging, as too much or too little lubricant on the eye will affect the image quality. 
	NOTE: The use of sterile lubricant gel is not recommended during OCT since it can interfere with imaging. If required, sterile lubricant gel can be used after the procedure. A contact lens can also be applied to ensure adequate moisture on the eye throughout the test. In our experience, a contact lens did not provide a marked improvement in image quality, but contact lenses do help reduce the risk of cornea drying during the imaging session.</li>
</ol>
3. Rodent OCT imaging
<ol>
	<li>Begin with one eye (OS or OD) and image the contralateral eye after.
	<ol>
		<li>Position the animal using the two rotational motions of the rodent alignment system, such that the gaze is horizontal and looking down the axis of the OCT Lens (Figure 1D).</li>
		<li>Use the OCT in Free Run mode to orient the retina for data collection. Use the Aim mode (by clicking the Aim button) initially for a continuous display of both horizontal and vertical B-scans in real time.</li>
		<li>Move the scan head closer to the eye until the retina is visible (as mouse and rat retina lenses are fixed-focus, moving the lens toward the eye focuses deeper into the retina). Then use the rodent alignment system to adjust the animal position up/down and swivel/twist to position the optic nerve head in the center, make the horizontal scan horizontal, and the vertical scan vertical (Figure 1A).</li>
		<li>Adjust the working distance such that the retinal image is flat and not curved.</li>
		<li>Adjust the reference arm position to keep the image near the top of the display window. Be careful not to push in too far or the eye image will flip back on itself.</li>
	</ol>
	</li>
	<li>Retinal imaging
	<ol>
		<li>For glaucoma, retinal degeneration, and diabetic retinopathy models: Define a volume scan that consists of 1000 x 100 x 1 (A scans x B scans x repeated B scans) for averaging. In rats, take a volume scan that is 3 x 3 mm. In mice, take a 1.5 x 1.5 mm volume scan.</li>
		<li>Center the optic nerve in the horizontal and the vertical access so that the volume scan is in the center. Take time to make sure the optic nerve head is at the center of the scan and straight along the nasal-temporal and superior-inferior axes (Figure 2). Scan and re-center to make sure it is exactly in the center, if needed. Repeat this scan as necessary until the optic nerve head is centered and aligned along both axes. Click the Snapshot button to take a photo. 
		NOTE: Some SD-OCT devices have the option of optically manipulating the curvature of the eye (e.g., the image is flattened) by adjusting the distance of the eye from the light source with the reference arm. We recommend flattening and centering the images when taking direct thickness measurements through the retinal layers to improve accuracy along the anterior-posterior direction.</li>
		<li>Click the Save button to save the image.</li>
		<li>Take a radial scan centered at the optic nerve head that is 1000 x 4 x 20 (A-scan x B-scan x repeated B-scans). Use repeated B scans to enhance image clarity of the eye or retina, which will help to interpret regions of the eye or layers of the retina during data analysis. 
		NOTE: Again, in rats this radial scan is 3 mm, while in mice the radial scan is 1.5 mm.</li>
		<li>Save the image.</li>
		<li>Repeat steps 3.1 through 3.2.5 in the contralateral eye.</li>
	</ol>
	</li>
	<li>Axial length measurements
	<ol>
		<li>For projects that involve imaging the whole eye, such as mouse myopia, take three scans of the entire eye and one retina scan for each eye. Choose a pre-set that consists of a radial scan that is 500 x 20 x 1 and encompasses the full diameter of the eye. 
		NOTE: This setting provides an image of the entire length of the mouse eye from the cornea to the choroid.</li>
		<li>Center the middle of the eye and retina in the field of view. Take three radial scans (whole eye scans): a linear B scan that is 1000 x 5 x 2 and two additional linear B scans of 1000 x 5 x 2 at the same location. Save the images.</li>
		<li>Afterward, if desired, zoom in and take a volume or rectangular scan (retina scan) similar to the description in 3.2 that consists of 1000 x 20 A scans x B scans. Save the volume scan.</li>
		<li>Repeat steps 3.3 through 3.3.3 in the contralateral eye. 
		NOTE: Axial length measurements are only possible on small eyes (mouse or smaller) since the imaging window of current systems is not large enough to capture a larger eye.</li>
	</ol>
	</li>
</ol>
4. Post-imaging steps
<ol>
	<li>Store saved data on a cloud, which is good practice for data management and allows for easy access for later analysis. Perform data analysis with custom software developed in a mathematical modeling program (Table of Materials).</li>
	<li>Remove the rodent from the rodent alignment system and give an intraperitoneal injection of atipamezole (1 mg/kg for rats and mice) to reverse the effects of the xylazine, so that the rodent will wake up more quickly.</li>
	<li>Allow rodents to recover on a heating pad on low heat. Give additional saline drops as needed. Return rodents to their home cage when they have regained full ambulation.</li>
	<li>Close program and turn off the OCT.</li>
</ol>
5. Post-processing of OCT images
<ol>
	<li>Process the images using custom software developed in a mathematical modeling program to suit specific OCT needs (e.g., measure the thickness of areas of interest by manually marking the images).</li>
	<li>Depending on the purpose of the image (mouse retina, rat retina, or myopia/axial length), use one of three different programs:
	<ol>
		<li>For processing the retina, select the OCT scans to load. First, define the center of the optic nerve head with a simple click.</li>
		<li>Watch as the program generates vertical lines defining distances on either side of the optic nerve head. Note that in the rat retina, these lines are at 0.5 mm and 1.2 mm away from the center of the optic nerve head, for a total of 4 vertical lines representing the nasal-temporal and inferior-superior axes of the eye depending on the radial B scan currently analyzed. 
		NOTE: In the mouse retina, these vertical lines are at 0.25 mm and 0.5 mm from the optic nerve head center.</li>
		<li>Delineate the following layers along each line: 
		The retinal nerve fiber layer (RNFL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), external limiting membrane (ELM), inner segments/outer segments (IS/OS), retinal pigment epithelium (RPE), and total retinal thickness. 
		NOTE: The radial scan does not typically have nasal/temporal and superior/inferior labels when it is opened. Scans may be created such that they have an n/t and s/I orientation, and those scans in particular are analyzed later.</li>
		<li>After an image has been delineated and the program closed, export these measurements into a spreadsheet software for data analysis.</li>
	</ol>
	</li>
	<li>Use these length and thickness values from step 5 to make comparisons between groups, for example, determining if there are regional differences (n/t/s/i), or longitudinal changes.</li>
	<li>For retinal measurements, first determine if there are any differences in the nasal-temporal and inferior-superior axis at the 0.5 mm and 1.2 mm distances. 
	NOTE: If differences in quadrants are not observed, the 0.5 mm and 1.2 mm measurements may be averaged together. This is a similar approach for the mouse retinal scans only at 0.25 mm and 0.5 mm.</li>
	<li>For myopia studies, use this program to assess the ocular parameters along the optical axis of the eye. Open the mathematical modeling program. First, select an image to load.
	<ol>
		<li>After loading the image, manually mark each scan (radial and B scans). Mark the anterior and posterior edges of the cornea, lens, vitreous chamber, and retina, so that the program will calculate corneal thickness, lens thickness, anterior and vitreous chamber depth, total retinal thickness, total axial length.</li>
		<li>After marking, exit the program which prompts a save menu. Save the delineated values in a spreadsheet software and average the three separate scans together.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Wojtkowski, 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=Ultrahigh-resolution,+high-speed,+Fourier+domain+optical+coherence+tomography+and+methods+for+dispersion+compensation.">Ultrahigh-resolution, high-speed, Fourier domain optical coherence tomography and methods for dispersion compensation.</a> Optics Express. 12 (11), 2404-2422 (2004).</li><li>Nassif, N., 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+high-resolution+video-rate+spectral-domain+optical+coherence+tomography+of+the+human+retina+and+optic+nerve.">In vivo high-resolution video-rate spectral-domain optical coherence tomography of the human retina and optic nerve.</a> Optics Express. 12 (3), 367-376 (2004).</li><li>Theelen, T., Teussink, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inspection+of+the+Human+Retina+by+Optical+Coherence+Tomography.">Inspection of the Human Retina by Optical Coherence Tomography.</a> Methods in Molecular Biology. 1715, 351-358 (2018).</li><li>Nakazawa, M., Hara, A., Ishiguro, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Coherence+Tomography+of+Animal+Models+of+Retinitis+Pigmentosa:+From+Animal+Studies+to+Clinical+Applications.">Optical Coherence Tomography of Animal Models of Retinitis Pigmentosa: From Animal Studies to Clinical Applications.</a> Biomed Research International. 2019, 8276140 (2019).</li><li>Drexler, W., 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=Ultrahigh-resolution+ophthalmic+optical+coherence+tomography.">Ultrahigh-resolution ophthalmic optical coherence tomography.</a> Nature Medicine. 7 (4), 502-507 (2001).</li><li>VanLeeuwen, J. E., 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=Altered+AMPA+receptor+expression+with+treadmill+exercise+in+the+1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned+mouse+model+of+basal+ganglia+injury.">Altered AMPA receptor expression with treadmill exercise in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse model of basal ganglia injury.</a> Journal of Neuroscience Research. 88 (3), 650-668 (2010).</li><li>Tian, J., 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=Performance+evaluation+of+automated+segmentation+software+on+optical+coherence+tomography+volume+data.">Performance evaluation of automated segmentation software on optical coherence tomography volume data.</a> Journal of Biophotonics. 9 (5), 478-489 (2016).</li><li>Kraus, M. F., 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=Motion+correction+in+optical+coherence+tomography+volumes+on+a+per+A-scan+basis+using+orthogonal+scan+patterns.">Motion correction in optical coherence tomography volumes on a per A-scan basis using orthogonal scan patterns.</a> Biomedical Optics Express. 3 (6), 1182-1199 (2012).</li><li>Boatright, J. H., 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=Tool+from+ancient+pharmacopoeia+prevents+vision+loss.">Tool from ancient pharmacopoeia prevents vision loss.</a> Molecular Vision. 12, 1706-1714 (2006).</li><li>Morrison, J. C., 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=A+rat+model+of+chronic+pressure-induced+optic+nerve+damage.">A rat model of chronic pressure-induced optic nerve damage.</a> Experimental Eye Research. 64 (1), 85-96 (1997).</li><li>Feola, A. J., 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=Menopause+exacerbates+visual+dysfunction+in+experimental+glaucoma.">Menopause exacerbates visual dysfunction in experimental glaucoma.</a> Experimental Eye Research. 186, 107706 (2019).</li><li>Goto, Y., Kakizaki, M., Masaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+spontaneous+diabetic+rats+by+repetition+of+selective+breeding.">Production of spontaneous diabetic rats by repetition of selective breeding.</a> The Tohoku Journal of Experimental Medicine. 119 (1), 85-90 (1976).</li><li>Allen, R. S., 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=Retinal+Deficits+Precede+Cognitive+and+Motor+Deficits+in+a+Rat+Model+of+Type+II+Diabetes.">Retinal Deficits Precede Cognitive and Motor Deficits in a Rat Model of Type II Diabetes.</a> Investigative Ophthalmolology &amp; Visual Science. 60 (1), 123-133 (2019).</li><li>Stone, R. 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=Altered+ocular+parameters+from+circadian+clock+gene+disruptions.">Altered ocular parameters from circadian clock gene disruptions.</a> PLoS One. 14 (6), 0217111 (2019).</li><li>Chakraborty, 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=Circadian+rhythms,+refractive+development,+and+myopia.">Circadian rhythms, refractive development, and myopia.</a> Ophthalmic &amp; Physiological Optics. 38 (3), 217-245 (2018).</li><li>Davies, E. C., 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=Retinal+ganglion+cell+layer+volumetric+assessment+by+spectral-domain+optical+coherence+tomography+in+multiple+sclerosis:+application+of+a+high-precision+manual+estimation+technique.">Retinal ganglion cell layer volumetric assessment by spectral-domain optical coherence tomography in multiple sclerosis: application of a high-precision manual estimation technique.</a> Journal of Neuro-ophthalmology. 31 (3), 260-264 (2011).</li><li>Carnevali, 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=Optical+coherence+tomography+angiography+analysis+of+retinal+vascular+plexuses+and+choriocapillaris+in+patients+with+type+1+diabetes+without+diabetic+retinopathy.">Optical coherence tomography angiography analysis of retinal vascular plexuses and choriocapillaris in patients with type 1 diabetes without diabetic retinopathy.</a> Acta Diabetologica. 54 (7), 695-702 (2017).</li><li>Springelkamp, H., 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=Population-based+evaluation+of+retinal+nerve+fiber+layer,+retinal+ganglion+cell+layer,+and+inner+plexiform+layer+as+a+diagnostic+tool+for+glaucoma.">Population-based evaluation of retinal nerve fiber layer, retinal ganglion cell layer, and inner plexiform layer as a diagnostic tool for glaucoma.</a> Investigative Ophthalmology &amp; Visual Science. 55 (12), 8428-8438 (2014).</li><li>Allen, R. S., 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=Long-Term+Functional+and+Structural+Consequences+of+Primary+Blast+Overpressure+to+the+Eye.">Long-Term Functional and Structural Consequences of Primary Blast Overpressure to the Eye.</a> Journal of Neurotrauma. 35 (17), 2104-2116 (2018).</li><li>Zhao, D., 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=Age-related+changes+in+the+response+of+retinal+structure,+function+and+blood+flow+to+pressure+modification+in+rats.">Age-related changes in the response of retinal structure, function and blood flow to pressure modification in rats.</a> Scientific Reports. 8 (1), 2947 (2018).</li><li>Schmucker, C., Schaeffel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+paraxial+schematic+eye+model+for+the+growing+C57BL/6+mouse.">A paraxial schematic eye model for the growing C57BL/6 mouse.</a> Vision Research. 44 (16), 1857-1867 (2004).</li><li>Aung, M. H., Kim, M. K., Olson, D. E., Thule, P. M., Pardue, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+visual+deficits+in+streptozotocin-induced+diabetic+long+evans+rats.">Early visual deficits in streptozotocin-induced diabetic long evans rats.</a> Investigative Ophthalmology &amp; Visual Science. 54 (2), 1370-1377 (2013).</li><li>Puzyeyeva, O., 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=High-Resolution+Optical+Coherence+Tomography+Retinal+Imaging:+A+Case+Series+Illustrating+Potential+and+Limitations.">High-Resolution Optical Coherence Tomography Retinal Imaging: A Case Series Illustrating Potential and Limitations.</a> Journal of Ophthalmology. 2011, 764183 (2011).</li><li>Liu, A. S., 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=Topography+and+pachymetry+maps+for+mouse+corneas+using+optical+coherence+tomography.">Topography and pachymetry maps for mouse corneas using optical coherence tomography.</a> Experimental Eye Research. 190, 107868 (2020).</li><li>Mohan, K., Kecova, H., Hernandez-Merino, E., Kardon, R. H., Harper, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cell+damage+in+an+experimental+rodent+model+of+blast-mediated+traumatic+brain+injury.">Retinal ganglion cell damage in an experimental rodent model of blast-mediated traumatic brain injury.</a> Investigative Ophthalmology &amp; Visual Science. 54 (5), 3440-3450 (2013).</li><li>Harper, M. 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=Blast-Mediated+Traumatic+Brain+Injury+Exacerbates+Retinal+Damage+and+Amyloidosis+in+the+APPswePSENd19e+Mouse+Model+of+Alzheimer's+Disease.">Blast-Mediated Traumatic Brain Injury Exacerbates Retinal Damage and Amyloidosis in the APPswePSENd19e Mouse Model of Alzheimer's Disease.</a> Investigative Ophthalmology Visual Science. 60 (7), 2716-2725 (2019).</li><li>Zhang, 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=Advanced+image+processing+for+optical+coherence+tomographic+angiography+of+macular+diseases.">Advanced image processing for optical coherence tomographic angiography of macular diseases.</a> Biomedical Optics Express. 6 (12), 4661-4675 (2015).</li><li>Muhlfriedel, 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=Optimized+Subretinal+Injection+Technique+for+Gene+Therapy+Approaches.">Optimized Subretinal Injection Technique for Gene Therapy Approaches.</a> Methods in Molecular Biology. 1834, 405-412 (2019).</li><li>Adekunle, A. N., 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=Integration+of+Perforated+Subretinal+Prostheses+With+Retinal+Tissue.">Integration of Perforated Subretinal Prostheses With Retinal Tissue.</a> Translational Vision Science &amp; Technology. 4 (4), 5 (2015).</li><li>Sajdak, B. S., 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=Noninvasive+imaging+of+the+tree+shrew+eye:+Wavefront+analysis+and+retinal+imaging+with+correlative+histology.">Noninvasive imaging of the tree shrew eye: Wavefront analysis and retinal imaging with correlative histology.</a> Experimental Eye Research. 185, 107683 (2019).</li><li>Dominik Fischer, 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=Detailed+functional+and+structural+characterization+of+a+macular+lesion+in+a+rhesus+macaque.">Detailed functional and structural characterization of a macular lesion in a rhesus macaque.</a> Documenta Ophthalmologica. 125 (3), 179-194 (2012).</li><li>Hagag, A. M., Gao, S. S., Jia, Y., Huang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+angiography:+Technical+principles+and+clinical+applications+in+ophthalmology.">Optical coherence tomography angiography: Technical principles and clinical applications in ophthalmology.</a> Taiwan Journal of Ophthalmology. 7 (3), 115-129 (2017).</li><li>Treister, A. D., 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=Prevalence+of+Subclinical+CNV+and+Choriocapillaris+Nonperfusion+in+Fellow+Eyes+of+Unilateral+Exudative+AMD+on+OCT+Angiography.">Prevalence of Subclinical CNV and Choriocapillaris Nonperfusion in Fellow Eyes of Unilateral Exudative AMD on OCT Angiography.</a> Translational Vision Science &amp; Technology. 7 (5), 19 (2018).</li></ol>

The authors have nothing to disclose.

High resolution imaging of ocular structures in vivo allows for the assessment of retinal and ocular changes over time. In this protocol, SD-OCT was demonstrated to capture differences in ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.
The most critical aspect when performing SD-OCT is obtaining a clear image of the retina or other ocular structure of interest. It is important to take time to make sure the retina is perfectly centered an...

Spectral-domain optical coherence tomography (SD-OCT) is a precise, high-resolution imaging modality that allows clinicians and researchers to examine ocular structures noninvasively. This imaging technique is based on interferometry to capture three-dimensional retinal images in vivo on a micrometer scale1,2. It has become one of the most frequently used imaging modalities in vision research and in the clinic due to the easy detection and accuracy of pathological features such as structural defects and/or thinning of retinal layers and subretinal fluid3. In research using animal models of vision-related disorders, SD-OCT has provided essential noninvasive analyses of relationships between structure and function and their histopathological origins4. Due to its resolution (up to 2-3 microns, depending on the depth into the eye5), SD-OCT has the capability to detect even small changes in retinal layer thickness. This type of analysis can provide essential information for disease progression and assess the efficacy of neuroprotective methods and treatments for vision-related disorders.
SD-OCT is a noninvasive alternative to examining structure histologically, and the two have been shown to be correlated6. While SD-OCT does not reach cellular resolution, it does allow for longitudinal studies in animals. This is advantageous because disease progression can be tracked in individual animals over time as opposed to having to euthanize animals at specific time points. As imaging techniques continue to improve, SD-OCT technology will also progress, providing enhanced image quality as well as the capability to assess biological processes such as retinal blood vessel function in fine detail. Even since its advent in 1991, SD-OCT technology has seen huge advances in resolution, speed, and sensitivity7.
The present study utilizes an SD-OCT system to quantify changes in retinal layers in rodent models of retinal degeneration, glaucoma, and diabetic retinopathy. The SD-OCT system used here is a Fourier-domain OCT-system that utilizes low-power, near-infrared light to acquire, process, and store depth-resolved images in real time. The SD-OCT system has extended depth-imaging capability in the 800 nm wavelength band, providing 8 mm depth and 4 &#181;m resolution. In Fourier domain detection, the interference signal between scattered light from the tissue and a reference path is Fourier transformed to construct axial scans and/or axial depth profiles of scattered intensity8. For the studies here, the OCT beam is scanned over the desired retinal structure while serially acquiring axial scans. Typically, a scan pattern acquires the two-dimensional grid (B-Scans) as a collection of linear one-dimensional scan lines (A-Scans), which correspond to 2D cross-sectional images using a raster scan pattern. For studies focused on myopia in mice, this system is also used to measure dimensions of ocular structures (e.g., cornea thickness, lens thickness, vitreous chamber depth, and axial length).
The current system allows users to design their own protocols, creating scans that can be tailored and selected based on the ocular structures of interest. The principal scans featured in these user defined protocols makes this imaging technique user-friendly. For image analyses, we have developed customized programming in a mathematic modeling program. SD-OCT is a powerful tool to non-invasively identify and quantify pathomorphological changes in ocular structures and monitor vision-related disease progression.

<table>
	<tbody>
		<tr>
			<td>1% tropicamide</td>
			<td>Sandoz</td>
			<td>Sandoz #6131403550; NDC- 24208-585-59</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% tetracaine</td>
			<td>Alcon</td>
			<td>NDC 0065-0741-12</td>
			<td></td>
		</tr>
		<tr>
			<td>AIM-RAS G3 120 V</td>
			<td>Leica Bioptigen</td>
			<td>90-AIMRAS-G3-120</td>
			<td>Specialized platform to hold the OCT Scanner Head for mice</td>
		</tr>
		<tr>
			<td>Celluvisc gel</td>
			<td>REFRESH CELLUVISC</td>
			<td>#4554; NDC-0023-4554-30</td>
			<td></td>
		</tr>
		<tr>
			<td>G3 18 mm Telecentric Lens</td>
			<td>Leica Bioptigen</td>
			<td>90-BORE-G3-18</td>
			<td></td>
		</tr>
		<tr>
			<td>G3 Mouse Lens</td>
			<td>Leica Bioptigen</td>
			<td>90-BORE-G3-M</td>
			<td></td>
		</tr>
		<tr>
			<td>G3 Rat Lens</td>
			<td>Leica Bioptigen</td>
			<td>90-BORE-G3-R</td>
			<td></td>
		</tr>
		<tr>
			<td>heating pad</td>
			<td>Fabrication</td>
			<td>11-1130</td>
			<td></td>
		</tr>
		<tr>
			<td>InVivoVue software</td>
			<td>Leica Bioptigen</td>
			<td></td>
			<td>Specialized software that pairs with the Leica Bioptigen SD-OCT system</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td>mathematical modeling program</td>
		</tr>
		<tr>
			<td>Mouse/Rat Kit</td>
			<td>Leica Bioptigen</td>
			<td>90-KIT-M/R</td>
			<td>Mouse/rat rodent alignment system</td>
		</tr>
		<tr>
			<td>saline</td>
			<td>ADDIPAK</td>
			<td>200-39</td>
			<td></td>
		</tr>
		<tr>
			<td>System Envisu R4300 VHR 120 V</td>
			<td>Leica Bioptigen</td>
			<td>90-R4300-V1-120</td>
			<td>SD-OCT system</td>
		</tr>
	</tbody>
</table>

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.

SD-OCT is considered successful if high quality images are obtained such that ocular dimensions can be reliably measured. Here, a variety of uses of SD-OCT are illustrated using models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.
In a light-induced retinal degeneration (LIRD) model, exposure to bright light (10,000 lux) induces degeneration of photoreceptor cells in the retina9. Representative SD-OCT images reveal a thinner outer nuclear layer, w...

Watch this Scientific Journal Video about In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography at JoVE.com

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

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 ...

in-vivo-structural-assessments-ocular-disease-rodent-models-using

Research

JoVE Journal

Bioengineering

2.7K Views.  Atlanta Veterans Affairs Medical Center. 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.

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

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 ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...