Name: switchable acoustic and optical resolution photoacoustic microscopy for in vivo small-animal blood vasculature imaging
Uploaded: 2017-06-26T15:00:00
Description: Watch this Scientific Journal Video about Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging at JoVE.com

The authors would like to acknowledge the financial support from a Tier 2 grant funded by the Ministry of Education in Singapore (ARC2/15: M4020238). The authors would also like to thank Mr. Chow Wai Hoong Bobby for the machine shop help.

All animal experiments were performed according to the approved regulations and guidelines of the Institutional Animal Care and Use Committee of Nanyang Technological University, Singapore (Animal Protocol Number ARF-SBS/NIE-A0263).
1. AR-OR-PAM System (Figure 1)
<ol>
	<li>System configuration: AR-PAM
	<ol>
		<li>Use a nanosecond tunable laser system consisting of a diode-pumped, solid-state Nd-YAG laser (532 nm) and a dye laser with a tunabilit...

<ol>
	<li>Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+imaging+and+characterization+of+the+microvasculature.">Photoacoustic imaging and characterization of the microvasculature.</a> J Biomed Opt. 15, 011101-01-011101-15 (2010).</li><li>Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+deeper+than+microscopy:+the+optical+imaging+frontier+in+biology.">Going deeper than microscopy: the optical imaging frontier in biology.</a> Nat Methods. 7 (8), 603-614 (2010).</li><li>Wang, L. V., Yao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+photoacoustic+tomography+in+the+life+sciences.">A practical guide to photoacoustic tomography in the life sciences.</a> Nat Methods. 13, 627-638 (2016).</li><li>Zhou, Y., Yao, J., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tutorial+on+photoacoustic+tomography.">Tutorial on photoacoustic tomography.</a> J Biomed Opt. 21 (6), 061007 (2016).</li><li>Upputuri, P. K., Sivasubramanian, K., Mark, C. S. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Developments+in+Vascular+Imaging+Techniques+in+Tissue+Engineering+and+Regenerative+Medicine.">Recent Developments in Vascular Imaging Techniques in Tissue Engineering and Regenerative Medicine.</a> BioMed Res Intl. 2015, (2015).</li><li>Yao, J., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+Brain+Imaging:+from+Microscopic+to+Macroscopic+Scales.">Photoacoustic Brain Imaging: from Microscopic to Macroscopic Scales.</a> Neurophotonics. 1 (1), 011003-1-011003-13 (2014).</li><li>Wang, L. V., Hu, S. <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:+In+Vivo+Imaging+from+Organelles+to+Organs.">Photoacoustic Tomography: In Vivo Imaging from Organelles to Organs.</a> Science. 335 (6075), 1458-1462 (2012).</li><li>Beard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+photoacoustic+imaging.">Biomedical photoacoustic imaging.</a> Interface Focus. 1 (4), 602-631 (2011).</li><li>Pan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+photoacoustic+imaging+of+angiogenesis+with+integrin-targeted+gold+nanobeacons.">Molecular photoacoustic imaging of angiogenesis with integrin-targeted gold nanobeacons.</a> FASEB J. 25 (3), 875-882 (2011).</li><li>Cai, X., Kim, C., Pramanik, M., Wang, L. V. <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+of+foreign+bodies+in+soft+biological+tissue.">Photoacoustic tomography of foreign bodies in soft biological tissue.</a> J Biomed Opt. 16 (4), 046017 (2011).</li><li>Pan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+infrared+photoacoustic+detection+of+sentinel+lymph+nodes+with+gold+nanobeacons.">Near infrared photoacoustic detection of sentinel lymph nodes with gold nanobeacons.</a> Biomaterials. 31 (14), 4088-4093 (2010).</li><li>Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+photoacoustic+microscopy+and+computed+tomography.">Multiscale photoacoustic microscopy and computed tomography.</a> Nat. Photon. 3 (9), 503-509 (2009).</li><li>Zhang, E. Z., Laufer, J. G., Pedley, R. B., Beard, P. C. <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+3D+photoacoustic+imaging+of+superficial+vascular+anatomy.">In vivo high-resolution 3D photoacoustic imaging of superficial vascular anatomy.</a> Phys. Med. Biol. 54 (4), 1035-1046 (2009).</li><li>Park, S., Lee, C., Kim, J., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+resolution+photoacoustic+microscopy.">Acoustic resolution photoacoustic microscopy.</a> Biomed.l Eng. Lett. 4 (3), 213-222 (2014).</li><li>Zhang, H. F., Maslov, K., Stoica, G., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+photoacoustic+microscopy+for+high-resolution+and+noninvasive+in+vivo+imaging.">Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging.</a> Nat. Biotechnol. 24 (7), 848-851 (2006).</li><li>Maslov, K., Stoica, G., Wang, L. V. <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+dark-field+reflection-mode+photoacoustic+microscopy.">In vivo dark-field reflection-mode photoacoustic microscopy.</a> Opt Lett. 30 (6), 625-627 (2005).</li><li>Strohm, E. M., Moore, M. J., Kolios, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Cell+Photoacoustic+Microscopy:+A+Review.">Single Cell Photoacoustic Microscopy: A Review.</a> IEEE J Sel Top Quantum Electron. 22 (3), 6801215 (2016).</li><li>Kim, J. Y., Lee, C., Park, K., Lim, G., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+optical-resolution+photoacoustic+microscopy+using+a+2-axis+water-proofing+MEMS+scanner.">Fast optical-resolution photoacoustic microscopy using a 2-axis water-proofing MEMS scanner.</a> Sci Rep. 5, 07932 (2015).</li><li>Matthews, T. P., Zhang, C., Yao, D. K., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+photoacoustic+microscopy+of+peripheral+nerves.">Label-free photoacoustic microscopy of peripheral nerves.</a> J Biomed Opt. 19 (1), 016004 (2014).</li><li>Hai, P., Yao, J., Maslov, K. I., Zhou, Y., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+optical-resolution+photoacoustic+microscopy.">Near-infrared optical-resolution photoacoustic microscopy.</a> Opt Lett. 39 (17), 5192-5195 (2014).</li><li>Danielli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+photoacoustic+nanoscopy.">Label-free photoacoustic nanoscopy.</a> J Biomed Opt. 19 (8), 086006 (2014).</li><li>Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflection-mode+submicron-resolution+in+vivo+photoacoustic+microscopy.">Reflection-mode submicron-resolution in vivo photoacoustic microscopy.</a> J Biomed Opt. 17 (2), 020501 (2012).</li><li>Hu, S., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-generation+optical-resolution+photoacoustic+microscopy+with+improved+sensitivity+and+speed.">Second-generation optical-resolution photoacoustic microscopy with improved sensitivity and speed.</a> Opt Lett. 36 (7), 1134-1136 (2011).</li><li>Maslov, K., Zhang, H. F., Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical-resolution+photoacoustic+microscopy+for+in+vivo+imaging+of+single+capillaries.">Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries.</a> Opt Lett. 33 (9), 929-931 (2008).</li><li>Upputuri, P. K., Krishnan, M., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsphere+enabled+sub-diffraction+limited+optical+resolution+photoacoustic+microscopy:+a+simulation+study.">Microsphere enabled sub-diffraction limited optical resolution photoacoustic microscopy: a simulation study.</a> J Biomed Opt. 22, 045001 (2017).</li><li>Upputuri, P. K., Wen, Z. B., Wu, Z., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+photoacoustic+microscopy+using+photonic+nanojets:+a+simulation+study.">Super-resolution photoacoustic microscopy using photonic nanojets: a simulation study.</a> J Biomed Opt. 19 (11), 116003 (2014).</li><li>Allen, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+fibre+lasers+as+excitation+sources+for+photoacoustic+tomography+and+microscopy+et+al.">Novel fibre lasers as excitation sources for photoacoustic tomography and microscopy et al.</a> Proc SPIE. , 97080W (2016).</li><li>Xing, W., Wang, L., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+optical-and+acoustic-resolution+photoacoustic+microscopy+based+on+an+optical+fiber+bundle.">Integrated optical-and acoustic-resolution photoacoustic microscopy based on an optical fiber bundle.</a> Opt Lett. 38 (1), 52-54 (2013).</li><li>Estrada, H., Turner, J., Kneipp, M., Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optoacoustic+brain+microscopy+with+hybrid+optical+and+acoustic+resolution.">Real-time optoacoustic brain microscopy with hybrid optical and acoustic resolution.</a> Laser Phys Lett. 11 (4), 045601 (2014).</li><li>Jeon, S., Kim, J., Kim, C. <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+switchable+optica-+and+acoustic+-+resolution+photoacoustic+microscopy.">In vivo switchable optica- and acoustic - resolution photoacoustic microscopy.</a> Proc SPIE. , 970845 (2016).</li><li>Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+integrated+reflection-mode+photoacoustic,+two-photon,+and+second+harmonic+generation+microscopy+in+vivo.">Fully integrated reflection-mode photoacoustic, two-photon, and second harmonic generation microscopy in vivo.</a> Sci Rep. 6, 32240 (2016).</li><li>Park, 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=Delay-multiply-and-sum-based+synthetic+aperture+focusing+in+Photoacoustic+microscopy.">Delay-multiply-and-sum-based synthetic aperture focusing in Photoacoustic microscopy.</a> J Biomed Opt. 21 (3), 036010-10 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANSI+Standard+Z136.1-2000.">ANSI Standard Z136.1-2000.</a> American National Standard for Safe Use of Lasers. , (2000).</li><li>Moothanchery, M., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+Characterization+of+a+Switchable+Acoustic+Resolution+and+Optical+Resolution+Photoacoustic+Microscopy+System.">Performance Characterization of a Switchable Acoustic Resolution and Optical Resolution Photoacoustic Microscopy System.</a> Sensors. 17 (2), 357 (2017).</li></ol>

In conclusion, a switchable AR and OR PAM system that can achieve both high-resolution imaging at lower imaging depths and lower-resolution imaging at higher imaging depths has been developed. The lateral resolution and imaging depth of the switchable system was determined. The advantages of this switchable PAM system include: (1) the high-resolution imaging using tight optical focusing; (2) the deep-tissue imaging using acoustic focusing; 3) the dark-field illumination for AR-PAM, which prevents strong PA signals from a...

High-resolution optical imaging modalities, such as optical coherence tomography, confocal microscopy, and multiphoton microscopy, have numerous benefits. However, the spatial resolution decreases significantly as the imaging depth increases. This is because of the diffuse nature of light transport in soft tissues1,2. The integration of optical excitation and ultrasound detection provides a solution to overcome the challenge of high-resolution optical imaging in deep tissues. Photoacoustic microscopy (PAM) is one such modality that can provide deeper imaging than other optical imaging modalities. It has been successfully applied to in vivo structural, functional, molecular, and cell imaging3,4,5,6,7,8,9,10,11,12,13 studies by combining the strong optical absorption contrast with the high spatial resolution from ultrasound.
In PAM, a short laser pulse irradiates the tissue/sample. The absorption of light by chromophores (e.g., melanin, hemoglobin, water etc.) results in a temperature increase, which in turn results in the production of pressure waves in the form of acoustics waves (photoacoustic waves). The generated photoacoustic waves can be detected by a wideband ultrasonic transducer outside the tissue boundary. Utilizing weak optical and tight acoustic focusing, deep-tissue imaging can be achieved in acoustic resolution photoacoustic microscopy (AR-PAM)14,15,16. In AR-PAM, a lateral resolution of 45 &#181;m and an imaging depth up to 3 mm have been demonstrated15. In order to resolve single capillaries (~5 &#181;m) acoustically, ultrasonic transducers operating at &#62;400 MHz central frequencies are required. At such high frequencies, the penetration depth is less than 100 &#181;m. The problem caused by tight acoustic focusing can be resolved using tight optical focusing. Optical resolution photoacoustic microscopy (OR-PAM) is capable of resolving single capillaries, or even a single cell17, and a lateral resolution of 0.5 &#181;m has been achieved18,19,20,21,22,23,24. The use of a photonic nanojet can help to achieve a resolution beyond the diffraction-limited resolution25,26. In OR-PAM, the penetration depth is limited due to light focusing, and it can image up to ~1.2 mm inside the biological tissue23. Therefore, AR-PAM can image deeper, but with a lower resolution, and OR-PAM can image with a very high resolution, but with limited imaging depth. The imaging speed of the AR and OR-PAM system mainly depends upon the pulse repetition rate of the laser source27.
Combining AR-PAM and OR-PAM will be of great benefit to applications that require both a high resolution and deeper imaging. Little effort has been made to combine these systems together. Usually, two different imaging scanners are used for imaging, which requires that the sample be moved between both systems, thus making it difficult to perform in vivo imaging. However, hybrid imaging with both AR and OR PAM enables imaging with scalable resolutions and depths. In one approach, an optical fiber bundle is used to deliver light for both the AR and OR PAM. In this approach, two separate lasers (a high-energy laser at 570 nm for the AR and a low-energy, high-repetition rate laser at 532 nm for the OR) are used, making the system inconvenient and expensive28. The OR-PAM laser wavelength is fixed, and many studies, such as on oxygen saturation, are not possible using this combined system. Comparative studies between AR and OR PAM are also not possible because of the difference in laser wavelengths between the AR and OR. Moreover, AR-PAM uses bright-field illumination; hence, strong photoacoustic signals from the skin surface limit the image quality. For this reason, the system cannot be used for many bioimaging applications. In another approach to perform AR and OR PAM, the optical and ultrasound focus is shifted, which makes the light focus and ultrasound focus unaligned. Thus, the image quality is not optimal29. Using this technique, the AR-PAM and OR-PAM can achieve only 139 &#181;m and 21-&#181;m resolutions, respectively, making it a poor-resolution system. Another approach, which includes changing the optical fiber and collimating optics, was reported to switch between AR and OR PAM, making the alignment process difficult30. In all of these cases, AR-PAM did not use dark-field illumination. The use of dark-field illumination can reduce the generation of strong photoacoustic signals from the skin surface. Therefore, deep-tissue imaging can be performed using ring-shaped illumination, as the detection sensitivity of deep photoacoustic signals will be higher compare to that of bright-field illumination.
This work reports a switchable AR and OR PAM (AR-OR-PAM) imaging system capable of both high-resolution imaging and low-resolution deep-tissue imaging of the same sample, using the same laser and scanner for both systems. The performance of the AR-OR-PAM system was characterized by determining the spatial resolution and imaging depth using phantom experiments. In vivo blood vasculature imaging was performed on a mouse ear to demonstrate its biological imaging capability.

<table border="0" cellpadding="0" cellspacing="0" width="939">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Q-switched Nd:YAG laser</td>
			<td style="width:232px;">Edgewave</td>
			<td style="width:177px;">BX80-2-L</td>
			<td style="width:227px;">Pump laser&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Credo-High Repetition Rate Dye Laser</td>
			<td>Spectra physics</td>
			<td style="width:177px;">CREDO-DYE-N</td>
			<td>Dye laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision Linear Stage</td>
			<td>Physik Instrumente</td>
			<td>PLS 85&#160;</td>
			<td>XY raster scanning stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Translation stage</td>
			<td>Physik Instrumente</td>
			<td>VT 80&#160;</td>
			<td>Confocal determine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounted Silicon photodiode</td>
			<td>Thorlabs</td>
			<td>SM05PD1A</td>
			<td>Triggering/Pulse variation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Motorized continuous Rotational stage&#160;</td>
			<td>Thorlabs</td>
			<td>CR1/M-Z7</td>
			<td>Diverting laser beam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounted Continuously Variable ND Filter</td>
			<td>Thorlabs</td>
			<td>NDC-50C-4M</td>
			<td>Intensity variable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiber Patch Cable</td>
			<td>Thorlabs</td>
			<td>M29L01</td>
			<td>Multimode fiber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope objective</td>
			<td>Newport</td>
			<td>M-10X</td>
			<td>Objective&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XY translating mount</td>
			<td>Thorlabs</td>
			<td>CXY1</td>
			<td>Translating mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Plano convex lens</td>
			<td>Thorlabs</td>
			<td>LA1951</td>
			<td>Collimating lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical lens&#160;</td>
			<td>Altechna</td>
			<td>APX-2-B254</td>
			<td>Ring shape beam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Translation stage</td>
			<td>Thorlabs</td>
			<td>CT1</td>
			<td>Translating stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Optical condenser</td>
			<td style="width:232px;">Home made</td>
			<td style="width:177px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasonic transducer</td>
			<td>Olympus-NDT</td>
			<td>V214-BB-RM</td>
			<td>50MHz transducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Plano concave lens</td>
			<td>Thorlabs</td>
			<td>LC4573</td>
			<td>Acoustic lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pulser/Receiver</td>
			<td>Olympus-NDT</td>
			<td>5073PR</td>
			<td>Pulse echo amplifier&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mounted standard iris</td>
			<td>Thorlabs</td>
			<td>ID12/M</td>
			<td>Beam shaping</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Plano convex lens</td>
			<td>Thorlabs</td>
			<td>LA4327</td>
			<td>Condenser lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mounted precision pinhole</td>
			<td>Thorlabs</td>
			<td>P50S</td>
			<td>Spatial filtering</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Single mode fiber patch cable</td>
			<td>Thorlabs</td>
			<td>P1-460B-FC-1</td>
			<td>Single mode fiber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fiber coupler</td>
			<td>Newport</td>
			<td>F-91-C1</td>
			<td>Single mode coupling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Achromatic doublet lens</td>
			<td style="width:232px;">Edmund Optics</td>
			<td>32-317</td>
			<td>Achromatic doublet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Protected silver elliptical mirror</td>
			<td>Thorlabs</td>
			<td>PFE10-P01</td>
			<td>Mirror</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Right angle kinematic mirror mount</td>
			<td>Thorlabs</td>
			<td>KCB1</td>
			<td>Mirror mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Z-Axis Translation Mount</td>
			<td>Thorlabs</td>
			<td>SM1Z</td>
			<td>z translator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Lens tube</td>
			<td>Thorlabs</td>
			<td>SM05L10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">UV Fused Silica Right-Angle Prism</td>
			<td>Thorlabs</td>
			<td>PS615</td>
			<td>Right angle prism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Rhomboid prism</td>
			<td style="width:232px;">Edmund Optics</td>
			<td>47-214</td>
			<td>Shear wave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethylpolysiloxane</td>
			<td>Sigma Aldrich</td>
			<td>DMPS1M</td>
			<td>Silicon oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Amplifier</td>
			<td>Mini Circuits</td>
			<td>ZFL-500LN</td>
			<td>Amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">16 bit high speed digitizer</td>
			<td>Spectrum</td>
			<td>M4i.4420</td>
			<td>Data acquisition card</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Oscilloscope</td>
			<td style="width:232px;">Agilent Technologies</td>
			<td style="width:177px;">DS06014A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mice&#160;</td>
			<td style="width:232px;">InVivos Pte.Ltd</td>
			<td style="width:177px;">ICR</td>
			<td>Animal model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ultrasound gel&#160;</td>
			<td>Progress/parker acquasonic gel</td>
			<td>PA-GEL-CLEA-5000</td>
			<td>Acoustic coupling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Water tank</td>
			<td style="width:232px;">Home made</td>
			<td style="width:177px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Translation stage</td>
			<td style="width:232px;">Homemade</td>
			<td style="width:177px;"></td>
			<td>Switching AR-OR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Gold nanoparticles</td>
			<td>Sigma Aldrich</td>
			<td align="right">742031</td>
			<td>Lateral resolution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sterile ocular ointment</td>
			<td style="width:232px;">Alcon</td>
			<td style="width:177px;">Duratears</td>
			<td>Animal imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1951 USAF resolution test target</td>
			<td style="width:232px;">Edmund Optics</td>
			<td align="right" style="width:177px;">38257</td>
			<td>Confocal alignment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Data acquisition software</td>
			<td style="width:232px;">National Instrument</td>
			<td style="width:177px;">Labview</td>
			<td>Home made software using Labview</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Image Processing software</td>
			<td style="width:232px;">Mathworks</td>
			<td style="width:177px;">Matlab</td>
			<td>Home made program using Matlab</td>
		</tr>
	</tbody>
</table>

switchable acoustic and optical resolution photoacoustic microscopy for in vivo small-animal blood vasculature imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

The schematic of the AR-OR-PAM system is shown in Figure 1. In this setup, all components were integrated and assembled in an optical cage setup. The use of a cage system makes the AR-OR-PAM scanning head compact and easily assembled, aligned, and integrated onto a single scanning stage.
Two-dimensional continuous raster scanning of the imaging head was used during image acquisition. The time-resolv...

Watch this Scientific Journal Video about Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging at JoVE.com

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

Research

JoVE Journal

Bioengineering

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A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

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