This work was supported by the generous support of the National Eye Institute 4K12EY022299 (YMP), Fight for Sight-International Retinal Research Foundation FFS GIA16002 (YMP), unrestricted departmental support from Research to Prevent Blindness, and the University of Michigan Department of Ophthalmology and Visual Sciences. This work utilized the Core Center for Vision Research funded by P30 EY007003 from the National Eye Institute.

Rabbits are a United States Department of Agriculture (USDA) covered species. Its use in biomedical research needs to follow strict regulations. All rabbit experiments were performed in accordance with the ARVO (The Association for Research in Vision and Ophthalmology) Statement for the Use of Animals in Ophthalmic and Vision Research, after approval of the laboratory animal protocol by the University Committee on Use and Care of Animals (UCUCA) of the University of Michigan (Protocol PRO00006486, PI Yannis Paulus).
1. System configuration
<ol>
	<li>Photoacoustic microscopy (PAM)
	<ol>
		<li>Use an optical parametric oscillator (OPO) laser pumped by a diode-pumped solid-state laser as the light source of the PAM. Select proper technical specifications, such as pulse repetition rate 1 kHz, pulse duration 3 - 6 ns, and tunable wavelength range 405 - 2600 nm.</li>
		<li>Reflect the emanating beam from the laser at 570 nm by two mirrors (M1 and M2), then pass it through a half-wave plate attenuator mounted on a motorized rotation stage, and finally focus, filter, and collimate it by a beam collimator (Figure 1). Optimize the design of the beam collimator. An example configuration of the beam collimator includes a focusing lens L1 (focal length 250 mm), a pinhole (diameter 50 &#181;m), and a collimating lens L2 (focal length 30 mm).</li>
		<li>Divide the collimated beam by a beam splitter (BS1) with a split ratio of 90/10 (reflection/transmission). Record the transmitted portion by a photodiode for pulse-to-pulse laser energy monitoring. Successively deflect the reflected portion by a mirror (M3) and a dichroic mirror (DM) and raster-scan it using a two-dimensional galvanometer. The galvanometer is a shared component with the spectral domain (SD)-OCT system (described below).</li>
		<li>Deliver the scanned beam through a telescope composed of a scan lens (focal length 36 mm) and an ophthalmic lens (OL, focal length 10 mm) and finally focus it on the fundus by the rabbit eye optics.</li>
		<li>Select a needle-shaped ultrasonic transducer with proper technical specifications, for example, center frequency 27 MHz, two-way &#8722;6 dB bandwidth 60%. Place it in contact with the conjunctiva off the central visual axis to capture excited photoacoustic signal.</li>
		<li>Amplify the signal using an ultrasonic amplifier (for example, gain 57 dB), filter it by a low-pass filter (for example, cutoff frequency 32 MHz), and digitize it by a high-speed digitizer at a sampling rate of 200 MS/s.</li>
		<li>Place a power meter above the rabbit eye and measure the laser pulse energy on the rabbit cornea to keep it below the ANSI safety limit 160 nJ at 570 nm38. Block the beam to avoid laser overexposure using a laser shutter controlled from Matlab through a synchronization electronics.</li>
		<li>Synchronize the laser, the galvanometer, and the digitizer through a data acquisition (DAQ) board. Program the software for system control and data acquisition in Matlab.</li>
	</ol>
	</li>
	<li>Spectral-domain optical coherence tomography (SD-OCT)
	<ol>
		<li>Adapt the SD-OCT system based on a commercially available system by adding an ophthalmic lens (OL) after the scan lens (SL) and a piece of dispersion compensation glass (DCG) in the reference arm (Figure 1). The modification enables that the OCT system can image the posterior segment of the rabbit eye.</li>
		<li>Use a zooming housing tube to adjust the length of the reference arm to ensure its match with the optical path length of the sample arm. Use an iris to control the intensity of retro-reflected reference light to ensure its match with back-scattered light intensity from the rabbit fundus to achieve maximum image contrast.</li>
		<li>Employ a charge-coupled device (CCD) camera encapsulated in the scan head for real-time visualization of rabbit fundus with an illumination light emitting diode (LED) as an external illumination source.</li>
	</ol>
	</li>
</ol>
2. System alignment
<ol>
	<li>Initialize the position of the galvanometer and align the OCT system by adjusting the screws of the fiber collimator mount and the beamsplitter cube. 
	NOTE: The step-by-step procedures are available in the manual of the commercially acquired OCT system and will not be covered here. This step is mainly to ensure correct alignments of the fiber collimator, the reference arm, and the scan lens to maximize the performance of the OCT system.</li>
	<li>Adjust the x, y, and z position of the pinhole around the focus of the focusing lens to spatially filter out the laser beam and maximally transmit laser energy. Check the heights of the laser beam before and after the pinhole using a height measurement tool to ensure that they are the same.</li>
	<li>Adjust the tilt, decenter, and z position of the collimating lens L2 to collimate the filtered beam. Ensure that the beam has approximately the same size and height when observed in the near field and the far field.</li>
	<li>Co-axially combine the PAM laser beam and the OCT light beam by tuning the tilts of the mirror and the DM. After this step, the PAM laser and OCT light should be fully coincident and scanning regions on the rabbit fundus are the same.</li>
	<li>Adjust the tilt and decenter of the OL lens to correctly align it in the optical path. Once done, the dual-modality system is ready for imaging. 
	NOTE: One can use the auto-collimation method to achieve this, i.e., checking the light back-reflected by the OL lens surface to make sure that it goes back along the same way as the incident light.</li>
</ol>
3. Rabbit preparation
<ol>
	<li>Take a New Zealand White rabbit from the animal facility and record individual information, such as body weight and animal number.</li>
	<li>Mount rabbit platforms, including the body support and the head support on the optical table below the imaging system. Put a water-circulating heating blanket on the body support and set the temperature of the circulating water to 38 &#176;C to help keep the body temperature of the rabbit warm for the duration of the experiment and recovery.</li>
	<li>Record the pre-procedure vitals, including overall animal state, mucous membrane color, heart rate, respiratory rate, and rectal body temperature. Anesthetize the rabbit with a mixture of ketamine (40 mg/kg) and xylazine (5 mg/kg) through intramuscular (IM) injection and record the usage of ketamine (Schedule III controlled substance). Confirm the anesthesia level by checking its heart rate, respiratory rate, and overall state.</li>
	<li>Dilate the rabbit pupils using one drop each of Tropicamide 1% ophthalmic and phenylephrine hydrochloride 2.5% ophthalmic.</li>
	<li>Use a speculum to hold the eyelids out of the way and apply a drop of eye lubricant to moisten the cornea. Instill a drop of topical Tetracaine 0.5% in the eye before the imaging procedure. 
	NOTE: For longer procedures or procedures with possible discomfort to the animal, give the rabbit a subcutaneous injection of meloxicam before the experiment to ensure animal comfort.</li>
</ol>
4. SD-OCT imaging
<ol>
	<li>Transfer the rabbit to the imaging platform of a clinical fundus camera and take 50 degree fundus, red free, and autofluorescence images before the OCT imaging session. This helps check optical transparency of the eye and recognize fundus vessel morphology and landmarks, such as the optic nerve and medullary ray retinal vasculature.</li>
	<li>Transfer the rabbit to the platform of the OCT system and adjust its posture to roughly position one of the eyes under the OL. Illuminate the eye using the LED light. 
	Note: To facilitate the clinical translation of the technique, the rabbit eyes are not stabilized using any other methods and the rabbit head is just put on the head support without any fixation.</li>
	<li>Open the OCT software and first check the CCD camera image of the fundus. Finely adjust the height and angle of the head support if necessary to ensure region of interests (ROIs), such as choroidal vessels and retinal vessels, are within the field of view (FOV) of the camera. 
	Note: If the rabbit is under a good anesthesia level, one sequential imaging session can last as long as 10 min without the need of re-adjusting the rabbit head.</li>
	<li>Draw a straight line to represent the OCT B-scan of interest and start scanning. Adjust the reference arm length to visualize the OCT image and optimize the dispersion compensation factor in the OCT software to get the sharpest images. 
	Note: When adjusting the reference arm length, two mirrored OCT images will appear one after the other. The correct image could be distinguished based on prior knowledge of the fundus anatomy.</li>
	<li>Set data acquisition parameters, such as number of pixels and averages, and save images.</li>
	<li>Observe the respiratory rate and heart rate of the rabbit to estimate the anesthesia level and animal comfort. For longer sessions, a one third dose of supplemental ketamine or inhaled isoflurane could be considered with endotracheal intubation, a V-gel, or a face mask to maintain the plane of anesthesia when necessary.</li>
	<li>Rinse the rabbit cornea with eyewash every 2 min during the experiment to prevent corneal surface dehydration and corneal superficial punctate epithelial keratopathy. Monitor and record animal vitals every 15 min.</li>
</ol>
5. PAM imaging
<ol>
	<li>Wear appropriate laser safety goggles and turn on the OPO laser.</li>
	<li>Start the PAM control software, tune the laser wavelength to one of the absorption peaks of the targeted chromophores (e.g., 570 nm for hemoglobin), initialize the position of the galvanometer, and monitor laser energy before the rabbit cornea to ensure that it is below the ANSI safety limit.</li>
	<li>Mount the ultrasonic transducer on a three-dimensional (3D) translation stage and position the transducer tip in contact with the rabbit conjunctiva pointing to the fundus. Use a drop of eye lubricant to better couple the transducer tip and rabbit conjunctiva.</li>
	<li>Turn on the LED illumination light and visualize the rabbit fundus through the Matlab software.</li>
	<li>Set the scanning ROI (either retinal vessels or choroidal vessels), including the center and the physical size. Open the laser shutter and start B-scan of the beam. By roughly align the transducer, one should be able to see detected photoacoustic signal on the oscilloscope. If not, slightly adjust the eye position to scan a different region of the cornea or switch to the other eye and repeat the above processes.</li>
	<li>Observe the detected photoacoustic signal on the oscilloscope and finely tune the transducer position to maximize signal intensity along the entire B-scan. 
	Note: Due to limited beam width, ultrasonic transducer usually has a small FOV41. This step determines the background modulation of final PAM images. Misalignment will lead to PAM images with heterogeneous background and degrade image quality greatly.</li>
	<li>Set data acquisition parameters. This includes the number of pixels (e.g., 256&#215;256 pixels), sampling rate (e.g., 200 MS/s), and delay time. Start data acquisition. The Matlab software will automatically open the shutter to pass the laser beam when started and close the shutter to block the beam when finished to avoid laser overexposure. 
	Note: Limited by the pulse repetition rate (1 kHz) of the laser, it takes about 1 min to finish the data acquisition of an image with 256 &#215; 256 pixels.</li>
	<li>Process the raw data and visualize the PAM image in two dimensions (2D) through maximum intensity projection (MIP)13 or in 3D through volumetric rendering38.</li>
	<li>Unmount the ultrasonic transducer, rinse the tip using deionized water, and put it back to the storage case.</li>
	<li>Transfer the rabbit to the fundus camera and re-examine the fundus. This step helps to check whether there are any morphological changes of the fundus after the imaging session.</li>
	<li>Rinse the rabbit cornea with eyewash every two min during the experiment to prevent corneal surface dehydration and keratopathy. Monitor and record animal vitals every 15 min. 
	Note: The PAM, OCT, and fundus imaging sessions take approximately 1 h.</li>
</ol>
6. Post imaging
<ol>
	<li>After fundus re-examination using the fundus camera, disconnect the V-gel if connected. Rinse the eye using eyewash, apply flurbiprofen ophthalmic and neomycin and polymyxin B sulfates and dexamethasone ophthalmic ointment, and close the eye.</li>
	<li>Transfer the rabbit with the water-circulating blanket to a recover chamber. Shield the box from light and wait until the rabbit naturally wakes up. During this period, monitor animal vitals every 15 min and keep the record and return a copy to the animal facility for recordkeeping.</li>
	<li>Once the rabbit wakes up and is active, alert and walking normally, transport it back to the animal facility. If an acute experiment is planned, euthanize the animal using euthanasia solution (e.g., Beuthanasia, 0.22 mL/kg, intravenous injection in the marginal ear vein) and dispose of the carcass.</li>
	<li>Turn off the software and the laser. Clean the optical bench.</li>
</ol>

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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=Photoacoustic+tomography+imaging+and+estimation+of+oxygen+saturation+of+hemoglobin+in+ocular+tissue+of+rabbits.">Photoacoustic tomography imaging and estimation of oxygen saturation of hemoglobin in ocular tissue of rabbits.</a> Exp Eye Res. 138, 153-158 (2015).</li><li>Tian, C., Zhang, W., Mordovanakis, A., Wang, X., Paulus, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+chorioretinal+imaging+in+living+rabbits+using+integrated+photoacoustic+microscopy+and+optical+coherence+tomography.">Noninvasive chorioretinal imaging in living rabbits using integrated photoacoustic microscopy and optical coherence tomography.</a> Opt Express. 25 (14), 15947-15955 (2017).</li><li>Laser Institute of America. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+National+Standard+for+Safe+Use+of+Lasers+ANSI+Z136.1+-+2007.">American National Standard for Safe Use of Lasers ANSI Z136.1 - 2007.</a> American National Standards Institute, Inc. , (2007).</li><li>Hughes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+schematic+eye+for+the+rabbit.">A schematic eye for the rabbit.</a> Vision Res. 12 (1), 123 (1972).</li><li>Ma, T., 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=Systematic+study+of+high-frequency+ultrasonic+transducer+design+for+laser-scanning+photoacoustic+ophthalmoscopy.">Systematic study of high-frequency ultrasonic transducer design for laser-scanning photoacoustic ophthalmoscopy.</a> J Biomed Opt. 19 (1), 016015 (2014).</li><li>Song, W., Wei, Q., Jiao, S., Zhang, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+photoacoustic+ophthalmoscopy+and+spectral-domain+optical+coherence+tomography.">Integrated photoacoustic ophthalmoscopy and spectral-domain optical coherence tomography.</a> J Vis Exp. (71), (2013).</li><li>Mattison, S., Kim, W., Park, J., Applegate, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+in+Optical+Coherence+Tomography.">Molecular Imaging in Optical Coherence Tomography.</a> Curr Mol Imaging. 3 (2), 88-105 (2014).</li></ol>

The authors have nothing to disclose.

An intact and regular tear film is essential for high-quality fundus images. An irregular and deteriorated tear films can significantly degrade image quality42. To preserve the integrity of the tear film and prevent corneal superficial punctate keratopathy, it is critical to lubricate the cornea using eyewash very frequently, approximately every two min. If there are any concerns regarding the opacity of the eye, use a slit lamp and fluorescein strips to check the cornea conditions.
<p class="...

Recent decades have witnessed the explosive development of the field of biomedical photoacoustic imaging1,2,3,4,5,6,7,8. Based on the energy conversion of light into sound, the emerging photoacoustic imaging can visualize biological samples at scales from organelles, cells, tissues, organs to small-animal whole body and can reveal its anatomical, functional, molecular, genetic, and metabolic information1,2,9,10,11,12. Photoacoustic imaging has found unique applications in a range of biomedical fields, such as cell biology13,14, vascular biology15,16, neurology17,18, oncology19,20,21,22, dermatology23, pharmacology24, and hematology25,26. Its application in ophthalmology, that is, photoacoustic ocular imaging, has attracted substantial interests from both scientists and clinicians and is currently under active investigation.
In contrast to routinely used ocular imaging technologies27, such as fluorescein angiography (FA) and indocyanine green angiography (ICGA) (based on fluorescence contrast), optical coherence tomography (OCT) (based on optical scattering contrast), and its derivative OCT angiography (based on motion contrast of red blood cells), photoacoustic ocular imaging uses optical absorption as the contrast mechanism. This is different from conventional ocular imaging technologies and provides a unique tool for studying optical absorption properties of the eye, which are usually associated with the pathophysiological status of ocular tissue28. To date, significant excellent work has been done in photoacoustic ocular imaging29,30,31,32,33,34,35,36,37, but these studies focus on the posterior segment of the eyes of small animals, such as rats and mice. The pioneering studies well demonstrate the feasibility of photoacoustic imaging in ophthalmology but there is still a long way to go towards clinical translation of the technology since eyeball sizes of rats and mice are much smaller (less than one-third) than that of humans. Due to the propagation of ultrasound waves over a significantly longer distances, signal intensity and image quality may greatly suffer when the technique is used for imaging the posterior segment of larger eyes.
Towards this goal, we recently reported the noninvasive, label-free chorioretinal imaging in living rabbits using integrated photoacoustic microscopy (PAM) and spectral-domain OCT (SD-OCT)38. The system has excellent performance and could visualize the retina and choroid of the eyes of larger animals based on endogenous absorption and scattering contrast of ocular tissue. Preliminary results in rabbits show that the PAM could noninvasively distinguish individual retinal and choroidal blood vessels using a laser exposure dose (~80 nJ) significantly below the American National Standards Institute (ANSI) safety limit (160 nJ) at 570 nm39; and the OCT could clearly resolve different retinal layers, the choroid, and the sclera. It is the very first demonstration of posterior segment imaging of larger animals using PAM and might be a major step towards clinical translation of the technology considering that the eyeball size of rabbits (18.1 mm)40 is almost 80% of the axial length of humans (23.9 mm).
In this work, we provide a detailed description of the dual-modality imaging system and experimental protocols used for noninvasive, label-free chorioretinal imaging in living rabbits and demonstrate the system performance through representative retinal and choroidal imaging results.

<table><tbody><tr><td>&lt;strong&gt;Dual-modality imaging system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>OPO laser</td><td>Ekspla (Vilnius, Lithuania)</td><td>NT-242</td><td></td></tr><tr><td>Beam attenuator</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>AHWP10M-600</td><td></td></tr><tr><td>Motorized rotation stage</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>PRM1/MZ8</td><td></td></tr><tr><td>Motorized rotation stage controller</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>TDC001</td><td></td></tr><tr><td>Focusing lens</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>AC254-250-B</td><td></td></tr><tr><td>Pinhole</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>P50S</td><td></td></tr><tr><td>Collimating lens</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>AC127-030-B</td><td></td></tr><tr><td>Photodiode</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>PDA36A&amp;nbsp;</td><td></td></tr><tr><td>Laser shutter</td><td>Vincent Associates Inc. (Toronto, Canada)</td><td>LS6S2T0</td><td></td></tr><tr><td>Laser shutter driver</td><td>Vincent Associates Inc. (Toronto, Canada)</td><td>VCM-D1</td><td></td></tr><tr><td>Dichroic mirror</td><td>Semrock, Inc. (Rochester, NY, USA)</td><td>Di03-R785-t3-25&amp;times;36</td><td></td></tr><tr><td>Scan lens</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>OCT-LK3-BB</td><td></td></tr><tr><td>Ophthalmic lens</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>AC080-010-B-ML</td><td></td></tr><tr><td>Ultrasonic transducer</td><td>Optosonic Inc. (Arcadia, CA, USA)</td><td>Custom</td><td></td></tr><tr><td>Amplifier</td><td>L3 Narda-MITEQ (Hauppauge, NY, USA)</td><td>AU-1647</td><td></td></tr><tr><td>Band-pass filter</td><td>Mini-Circuits (Brooklyn, NY, USA)</td><td>BLP-30+</td><td></td></tr><tr><td>Digitizer</td><td>DynamicSignals LLC (Lockport, IL, USA)</td><td>PX1500-4&amp;nbsp;</td><td></td></tr><tr><td>Synchronization electronics</td><td>National Instruments Corporation (Austin, TX, USA)</td><td>USB-6353</td><td></td></tr><tr><td>OCT module</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>Ganymede-II-HR</td><td></td></tr><tr><td>Dispersion compensation glass</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>LSM03DC</td><td></td></tr><tr><td>Illumination LED light</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>MCWHF2&amp;nbsp;</td><td></td></tr><tr><td>Power meter</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>S121C&amp;nbsp;</td><td></td></tr><tr><td>Power meter interface</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>PM100USB&amp;nbsp;</td><td></td></tr><tr><td>Height measurement tool&amp;nbsp;</td><td>Thorlabs, Inc. (Newton, NJ, USA)</td><td>BHM1</td><td></td></tr><tr><td>Fundus camera</td><td>Topcon Corporation (Tokyo, Japan)&amp;nbsp;</td><td>TRC 50EX</td><td></td></tr><tr><td>Matlab</td><td>MathWorks (Natick, MA, USA)</td><td>2017a</td><td></td></tr><tr><td>Oscilloscope</td><td>Teledyne LeCroy (Chestnut Ridge, NY, USA)</td><td>WaveJet 354T</td><td></td></tr><tr><td>&lt;strong&gt;Animal experiment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Water-circulating blanket</td><td>Stryker Corporation (Kalamazoo, MI, USA)</td><td>TP-700</td><td></td></tr><tr><td>Ketamine hydrochloride injection</td><td>Par pharmaceutical, Inc. (Woodcliff Lake, NJ, USA)</td><td>NDC code 42023-115-10</td><td></td></tr><tr><td>Xylazine hydrochloride</td><td>VetOne (Boise, ID, USA)</td><td>NDC code 13985-704-10</td><td></td></tr><tr><td>Tropicamide ophthalmic</td><td>Akorn Pharmaceuticals Inc. (Lake Forest, IL, USA)</td><td>NDC code 17478-102-12</td><td></td></tr><tr><td>Phenylephrine hydrochloride ophthalmic</td><td>Paragon BioTeck, Inc. (Portland, OR, USA)</td><td>NDC code 42702-102-15</td><td></td></tr><tr><td>Eye lubricant</td><td>Hub Pharmaceuticals LLC (Rancho Cucamonga, CA, USA)</td><td>NDC code 17238-610-15</td><td></td></tr><tr><td>Eyewash</td><td>Altaire Pharmaceuticals, Inc. (Aquebogue, NY, USA)</td><td>NDC code 59390-175-18</td><td></td></tr><tr><td>Tetracaine hydrochloride ophthalmic solution</td><td>Bausch &amp;amp; Lomb, Inc. (Rochester, NY, USA)</td><td>NDC code 24208-920-64</td><td></td></tr><tr><td>Flurbiprofen sodium ophthalmic solution</td><td>Bausch &amp;amp; Lomb, Inc. (Rochester, NY, USA)</td><td>NDC code 24208-314-25</td><td></td></tr><tr><td>Neomycin and Polymyxin B Sulfates and Dexamethasone Ophthalmic Ointment</td><td>Bausch &amp;amp; Lomb, Inc. (Rochester, NY, USA)</td><td>NDC code 24208-795-35</td><td></td></tr><tr><td>Meloxicam injection</td><td>Henry Schein Inc. (Queens, NY, USA)</td><td>NDC code 11695-6925-1</td><td></td></tr></tbody></table>

novel photoacoustic microscopy and optical coherence tomography dual-modality chorioretinal imaging in living rabbit eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

The dual-modality imaging system and experimental protocol have been successfully tested in the authors&#39; laboratory using four New Zealand White rabbits. The following showcases some representative results.
Figure 1&#160;shows the schematic of the PAM and SD-OCT dual-modality imaging system. It is composed of the following modules: photoacoustic light source, variable laser attenuator, beam coll...

Watch this Scientific Journal Video about Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes at JoVE.com

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

novel-photoacoustic-microscopy-optical-coherence-tomography-dual

Research

JoVE Journal

Engineering

10.8K Views.  University of Michigan. Dit manuscript beschrijft de nieuwe instelling en werkwijze van een photoacoustic microscopie en optische coherentie tomografie dual-modaliteit systeem voor noninvasive, etiket-vrije chorioretinal beeldvorming van grotere dieren, zoals konijnen.

Video: Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

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 ...

Bioengineering

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 ...

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 ...

Neuroscience

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 ...

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 ...

Medicine

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

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 ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Biology

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

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