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>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Amplifier</td>
			<td style="width:279px;">L3 Narda-MITEQ (Hauppauge, NY, USA)</td>
			<td style="width:157px;">AU-1647</td>
			<td></td>
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		<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">
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			<td style="width:279px;">DynamicSignals LLC (Lockport, IL, USA)</td>
			<td style="width:157px;">PX1500-4&#160;</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Synchronization electronics</td>
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			<td style="width:157px;">USB-6353</td>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Dispersion compensation glass</td>
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			<td style="width:157px;">LSM03DC</td>
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		<tr height="21">
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			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
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		<tr height="21">
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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>
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		<tr height="21">
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			<td style="width:279px;">Thorlabs, Inc. (Newton, NJ, USA)</td>
			<td>BHM1</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Fundus camera</td>
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			<td>TRC 50EX</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Matlab</td>
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			<td style="width:157px;">2017a</td>
			<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>
			<td style="width:279px;">Stryker Corporation (Kalamazoo, MI, USA)</td>
			<td style="width:157px;">TP-700</td>
			<td></td>
		</tr>
		<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>
			<td>VetOne (Boise, ID, USA)</td>
			<td style="width:157px;">NDC code 13985-704-10</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
			<td></td>
		</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 ...

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