Name: novel photoacoustic microscopy and optical coherence tomography dual-modality chorioretinal imaging in living rabbit eyes
Uploaded: 2018-02-08T15:00:00
Description: Watch this Scientific Journal Video about Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes at JoVE.com

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).
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<ol>
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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.
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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 border="0" cellpadding="0" cellspacing="0" style="width:824px;" width="822">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Dual-modality imaging system</td>
			<td style="width:279px;"></td>
			<td style="width:157px;"></td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">OPO laser</td>
			<td style="width:279px;">Ekspla (Vilnius, Lithuania)</td>
			<td style="width:157px;">NT-242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Beam attenuator</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">AHWP10M-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Motorized rotation stage</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">PRM1/MZ8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Motorized rotation stage controller</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">TDC001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Focusing lens</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">AC254-250-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Pinhole</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">P50S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Collimating lens</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">AC127-030-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Photodiode</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">PDA36A&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Laser shutter</td>
			<td style="width:279px;">Vincent Associates Inc. (Toronto, Canada)</td>
			<td style="width:157px;">LS6S2T0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Laser shutter driver</td>
			<td style="width:279px;">Vincent Associates Inc. (Toronto, Canada)</td>
			<td style="width:157px;">VCM-D1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Dichroic mirror</td>
			<td style="width:279px;">Semrock, Inc. (Rochester, NY, USA)</td>
			<td style="width:157px;">Di03-R785-t3-25&#215;36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Scan lens</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">OCT-LK3-BB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Ophthalmic lens</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">AC080-010-B-ML</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Ultrasonic transducer</td>
			<td style="width:279px;">Optosonic Inc. (Arcadia, CA, USA)</td>
			<td style="width:157px;">Custom</td>
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		<tr height="21">
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			<td style="width:279px;">L3 Narda-MITEQ (Hauppauge, NY, USA)</td>
			<td style="width:157px;">AU-1647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Band-pass filter</td>
			<td style="width:279px;">Mini-Circuits (Brooklyn, NY, USA)</td>
			<td style="width:157px;">BLP-30+</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Digitizer</td>
			<td style="width:279px;">DynamicSignals LLC (Lockport, IL, USA)</td>
			<td style="width:157px;">PX1500-4&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Synchronization electronics</td>
			<td style="width:279px;">National Instruments Corporation (Austin, TX, USA)</td>
			<td style="width:157px;">USB-6353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">OCT module</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">Ganymede-II-HR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Dispersion compensation glass</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">LSM03DC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Illumination LED light</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td style="width:157px;">MCWHF2&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Power meter</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Power meter interface</td>
			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td>PM100USB&#160;</td>
			<td></td>
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		<tr height="21">
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			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td>BHM1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Fundus camera</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Matlab</td>
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			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Oscilloscope</td>
			<td style="width:279px;">Teledyne LeCroy (Chestnut Ridge, NY, USA)</td>
			<td style="width:157px;">WaveJet 354T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Animal experiment</td>
			<td style="width:279px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Water-circulating blanket</td>
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			<td style="width:157px;">TP-700</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Ketamine hydrochloride injection</td>
			<td style="width:279px;">Par pharmaceutical, Inc. (Woodcliff Lake, NJ, USA)</td>
			<td style="width:157px;">NDC code 42023-115-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Xylazine hydrochloride</td>
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			<td style="width:157px;">NDC code 13985-704-10</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Tropicamide ophthalmic</td>
			<td style="width:279px;">Akorn Pharmaceuticals Inc. (Lake Forest, IL, USA)</td>
			<td style="width:157px;">NDC code 17478-102-12</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Phenylephrine hydrochloride ophthalmic</td>
			<td style="width:279px;">Paragon BioTeck, Inc. (Portland, OR, USA)</td>
			<td style="width:157px;">NDC code 42702-102-15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Eye lubricant</td>
			<td style="width:279px;">Hub Pharmaceuticals LLC (Rancho Cucamonga, CA, USA)</td>
			<td style="width:157px;">NDC code 17238-610-15</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Eyewash</td>
			<td style="width:279px;">Altaire Pharmaceuticals, Inc. (Aquebogue, NY, USA)</td>
			<td style="width:157px;">NDC code 59390-175-18</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Tetracaine hydrochloride ophthalmic solution</td>
			<td style="width:279px;">Bausch &#38; Lomb, Inc. (Rochester, NY, USA)</td>
			<td style="width:157px;">NDC code 24208-920-64</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Flurbiprofen sodium ophthalmic solution</td>
			<td style="width:279px;">Bausch &#38; Lomb, Inc. (Rochester, NY, USA)</td>
			<td style="width:157px;">NDC code 24208-314-25</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:229px;">Neomycin and Polymyxin B Sulfates and Dexamethasone Ophthalmic Ointment</td>
			<td style="width:279px;">Bausch &#38; Lomb, Inc. (Rochester, NY, USA)</td>
			<td style="width:157px;">NDC code 24208-795-35</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Meloxicam injection</td>
			<td style="width:279px;">Henry Schein Inc. (Queens, NY, USA)</td>
			<td style="width:157px;">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 ...

The overall goal of this novel imaging setup and operating procedure is to perform photoacoustic microscopy and optical coherence tomography dual-modality for non-invasive, label-free chorioretinal imaging of the eyes of larger mammals, such as rabbits. This method can help answer key questions in ophthalmic imaging about the imaging depth, resolution, and clinical translation required to discern oxygen saturation, blood distribution, and melanin in larger animals. The main advantage of this technique is that it allows noninvasive visualization of ocular tissue for the acquisition of photoacoustic microscopy`and optical coherence tomography dual-modality images.To begin, select an optical parametric oscillator laser pumped by a diode pump solid state laser as the light source for the photoacoustic microscope and use a beam splitter to divide the column aided beam with a split ratio of 19 reflection to 10 transmission. Successively deflect the reflected portion by the M3 mirror and dichroic mirror and use a two-dimensional galvanometer to raster scan the reflection. Deliver the scanned beam through a telescope composed of a scanned lens with a focal length of 36 millimeters and an ophthalmic lens with a focal length of 10 millimeters and focus the beam on the fundus by the rabbit eye optics.Before beginning the imaging procedure, please a water circulating heating blanket on the body support and set the temperature of the circulating water to 38 degree Celsius. Record the pre procedure vitals and confirm the appropriate level of sedation by the rabbit's heart and respiratory rates. Dilate the rabbit pupils with one drop each of tropicamide 1%ophthalmic and phenylephrine hydrochloride 2.5%ophthalmic.Use an ultrasonic amplifier to amplify the signal and filter the signal by a low pass filter. Place a power meter above the rabbit eye and measure the laser pulse energy on the rabbit cornea to keep it below the American National Standards Institute safety limit. To enable the optical coherence tomography or OCT system to image the posterior segment of the rabbit eye, add an ophthalmic lens after the scan lens.Use a zooming housing tube to adjust the length of the reference arm to confirm its match with the optical path length of the sample arm. For spectral domain OCT imaging, place the rabbit on to the imaging platform of a clinical fundus camera and obtain 50 degree fundus red free and autofluoroscence images. Next, transfer the animal to the platform of the OCT system with one eye under the ophthalmic lens.Illuminate the eye using the LED light and open the OCT software. Check the CCD camera image of the fundus and draw a straight line in the software to represent the OCT B scan of interest. Begin scanning adjusting the reference arm length to visualize the OCT image and to optimize the dispersion compensation factor in the OCT software to get the sharpest images.Then set data, the appropriate data acquisition parameters and save the images. For photoacoustic microscope imaging, turn on the optical parametric oscillator laser. Start the photoacoustic microscope control software and tune the lase wavelength to one of the absorption peaks of the targeted chromaforce.Initialize the position of the galvanometer and monitor the laser energy before the rabbit cornea to confirm that it is below the American National Standard Institute Safety Limit. Map the ultrasonic transducer on a three dimensional translation stage, and place the transducer tip in contact with the rabbit conjunctiva so that the tip is pointing to the fundus. Use a drop of eye lubricant to better couple the transducer tip in the rabbit conjunctiva and turn on the LED illumination light.Visualize the rabbit fundus through the MatLab software and set the scanning region of interest including the center and the physical size of the region. Open the laser shutter and start B scan of the beam observing the detected photoacoustic signal on the oscilloscope and finally tuning the transducer position to maximize the signal intensity along the entire B scan as necessary. Then set the data acquisition parameters process the raw data and visualize the photoacoustic microscope image in two dimensions.At the end of the imaging session transfer the rabbit back to the fundus camera to examine the fundus for any post imaging morphological changes and rinse the rabbit cornea with eye wash. Apply flurbiprofen ophthalmic, neomycin and polymyxin b sulfates and dexamethasone ophthalmic ointment to the cornea and close the eye. Then transfer the rabbit and the water circulating blanket into a recovery chamber with monitoring until full recovery.In this representative fundus photograph, the choroidal vessels are spread over most parts of the rabbit funds, while the retinal vessels are confined within the medullary ray. Here a typical photoacoustic microscopy image of the choroidal vasculature within the fundus is shown with the choroidal vessels blood vessels delineated at high lateral resolution. In this OCT B scan image, the retina, choroid, and sclera are visualized with a high axial resolution with the choroidal vessels observed below the retinal pigment epithelium layer.Using the dual-modality imaging system 2D maximum intensity projection and 3D volumetric rendering of retinal vessels can be obtained by photoacoustic imaging. Orthogonal slices of the 3D image demonstrate how photoacoustic microscopy can also be used to visualize individual retinal vessels which lie above the retinal pigment epithelium layer and to confirm the different depths at which the retinal and choroidal vessels are located. Here an OCT B scan image shows individual retinal vessels.And the nerve fiber layer confirming the photoacoustic microscopy imaging results. Once mastered, this technique can be completed in approximately one hour if it is performed properly via to remember to set the laser irradiance at low ANLI setting limits to revive lubricants once eye rinse to avoid dehydration and to monitor the rabbit condition every 15 minutes. Following the procedure, automated slide immunohistochemistry can be performed to answer additional question about confirming choroidal neovascularization development and to amplify the volume of the neovascularization in rabbit.After its development this technique paved the way for researchers in the field of ophthalmology to explore choroidal diseases in rabbit bearing disease models including choroidal neovascularization models. After watching this video, you should have a good understanding of how to detect single chorioretinal blood vessels in rabbit eyes using a novel multimodal molecular imaging system for early stage diagnosis of wet age related macular degeneration. Don't forget this.Working with glass fall laser and animal can be extremely hazardous and that precaution such as wearing goggle and lab coats should always be taken while performing this procedure.

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