The authors would like acknowledge the financial support from National Natural Science Foundation of China (61822505; 11774101; 61627827; 81630046), The Science and Technology Planning Project of Guangdong Province, China (2015B020233016), and The Science and Technology Program of Guangzhou (No. 2019020001).

All animal experiments were performed in compliance with guidelines provided by the institutional animal care and use committee of South China Normal University, Guangzhou, China.
1. System Setup
<ol>
	<li>Optical path (Figure 1)
	<ol>
		<li>Use a 532 nm pulse laser as the system laser source. Set the repetition rate of the laser to 10 kHz, the output energy to 100% and the trigger setting to external trigger using a user-defined program.</li>
		<li>Couple the laser beam to single-mode fiber (SMF) via an optical fiber coupler (FC1). Collimate the laser beam using an optical fiber collimator (FC2) on a two-dimensional motorized stage (Motor, maximum speed: 20 mm/s).</li>
		<li>Deflect the laser beam using a two-axis galvanometer scanner (Galva). Use a moveable mirror (M1) to reflect the beam. Focus the beam through a 4&#215; objective lens (OL, Numerical aperture: 0.1).</li>
		<li>Use an XY translator mount (TM) to fix the self-made hollow ultrasonic transducer (UT, Central frequency: 25 MHz; Bandwidth: more than 90%; Center hole: 3 mm) on the bottom of the OL12. Pass the focused beam through the center hole of the ultrasonic transducer.</li>
	</ol>
	</li>
	<li>Scanning path
	<ol>
		<li>Lock the Galva using a field-programmable gate array (FPGA 2) during WIM. Set the appropriate scanning range and scanning speed by a user-defined program.</li>
		<li>Lock the Motor using a field-programmable gate array (FPGA 1) during RIM. Set the scanning frequency and number of scanning points using FPGA 2. Use a user-defined program to control the start and stop scanning.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Use a 50-dB amplifier (AMP) to amplify the PA signal. Digitize the signal by the data acquisition card (DAQ). Obtain the trigger signal through FPGA 1 or FPGA 2.</li>
		<li>Use a graphics processing unit (GPU) to process data and display images in parallel13.</li>
	</ol>
	</li>
	<li>CCD imaging system
	<ol>
		<li>Use a ring-shaped white LED (Color temperature: 6500 K; Illuminance: 40000 lux; Diameter: 7.5 cm) as a lighting source. Remove M1, use a fixed mirror (M2) to reflect the light.</li>
		<li>Record the images using a CCD camera (6.3 million pixels) on the PA imaging system. Display the images with a display software.</li>
	</ol>
	</li>
</ol>
2. System alignment
<ol>
	<li>Select a water tank (10 cm &#215; 10 cm &#215; 4.4 cm; bottom window: 3 cm &#215; 3 cm). Cover the entire water tank using a polyethylene membrane (membrane thick: 10 &#956;m). Add sufficient ultrapure water.</li>
	<li>Place the water tank on the working stage.</li>
	<li>Turn on the laser switch. Select the laser control program. Preheat for 5 min. Press the &#8220;ON&#8221; button on the pumping switch. Set the laser parameters as per step 1.1.1. Open the baffle of the laser.</li>
	<li>Select the A-line collected program. Press the &#8220;Start&#8221; button to capture the single point signal and display amplitude and spectrum of the current A-line signal.</li>
	<li>Place a blade at the bottom of the water tank. Immerse the bottom part of UT in the water tank for acoustic coupling. Avoid bubbles in the bottom part of UT.</li>
	<li>Adjust the position of Galva, adjust XY translator between UT and OL to avoid oscillation signal, and make sure this is confocal.</li>
	<li>Adjust the height of the working stage to maximize the amplitude of the signal, and determine the focus position.</li>
</ol>
3. Animal experiment
<ol>
	<li>Use a 5&#8210;6 weeks old BALB/c mouse with a bodyweight of 20&#8210;30 g.</li>
	<li>Anesthetize the animal using urethane (1 g/kg) injected intraperitoneally before the experiment.&#160;</li>
	<li>Conduct transition between WIM and RIM.
	<ol>
		<li>Use a planar ultrasonic transducer. Shave the fur on the back of the mouse using a trimmer and depilatory cream. Place the mouse on the holder (8 cm &#215; 2.8 cm &#215; 2 cm) in prone position.</li>
		<li>Allow the imaging region to be in contact with the polyethylene membrane using ultrasound gel. Avoid bubbles in the contact part.</li>
		<li>Place the holder on the working stage for acoustic coupling. Follow steps 2.3&#8210;2.4 to start the laser and collect A-line signal. Follow steps 2.6&#8210;2.7 to align. Press &#8220;Stop&#8221; to end the collection after alignment.</li>
		<li>Select the WIM program. Name the newly created folder. Set the scanning parameter at to 20 mm/s in the &#8220;Scanning Speed&#8221; tab, &#8220;20 mm*20 mm&#8221; in the &#8220;Scanning Area&#8221; tab, and &#8220;20&#8221; in the &#8220;Step&#8221; tab. Click the Collect button to start scanning.</li>
		<li>Click the Stop button to end scanning after acquisition. Click Return to Zero to bring the Motor to zero. Close the laser baffle. Set the trigger setting to internal trigger. Press the OFF button for pumping switch.</li>
		<li>Replace WIM trigger as RIM trigger and connect it to the external laser trigger. Press the ON button for pumping switch. Set the trigger setting to the external trigger. Click the Exit button to exit the WIM program.</li>
		<li>Use step 2.4 to collect the A-line signal. Open the laser baffle. Follow steps 2.6&#8210;2.7 to align. Press Stop to end collection after alignment.</li>
		<li>Select the RIM program. Name the newly created folder. Click the Collect button to start scanning.</li>
		<li>Click the Stop button to end scanning after completing acquisition. Click the Exit button to exit the RIM program.</li>
		<li>Euthanize the animal using cervical dislocation at the conclusion of the imaging.</li>
	</ol>
	</li>
	<li>Conduct WIM of vascular visualization.
	<ol>
		<li>Use a focused ultrasonic transducer (Central frequency: 25 MHz; Bandwidth: more than 90%; Focal length: 8 mm). Remove the hair of mice ear or scalp.
		<ol>
			<li>Use a scalpel to make a small incision on the lateral side of the cranial temporal top of the mouse (depth to the skull). Use ophthalmic scissors to start from this incision. Cut the scalp around the outer side of the skull. Compress the bleeding point to stop bleeding. Wash the wound with normal saline. Place the mouse on the holder.</li>
		</ol>
		</li>
		<li>Allow the imaging region to be in contact with the polyethylene membrane using ultrasound gel. Avoid bubbles in the contact region (Supplementary Figure 1).</li>
		<li>Place the holder on the work stage for acoustic coupling. Use steps 2.3&#8210;2.4 to open laser and collect A-line signal. Use step 2.6&#8210;2.7 to align. Press Stop to end collection after alignment.</li>
		<li>Select WIM program. Name the newly created folder. Set the scanning parameter to &#8220;10 mm/s&#8221; in the &#8220;Scanning Speed&#8221; tab, &#8220;10 mm*10 mm&#8221; under the &#8220;Scanning Area&#8221; tab, and &#8220;10&#8221; in the &#8220;Step&#8221; tab. Click the Collect button to start scanning.</li>
		<li>Click the Stop button to end scanning after completing acquisition. Click the Return to Zero to make the Motor return to zero. Click the Exit button to exit the WIM program.</li>
		<li>Euthanize the animal at the conclusion of the procedure while the animal is still under anesthesia.</li>
	</ol>
	</li>
	<li>Conduct RIM for dynamic monitoring of small animals.
	<ol>
		<li>Shave the hair of the mouse abdomen. Place the mouse on the holder in supine position.</li>
		<li>Allow the imaging region to be in contact with the polyethylene membrane using ultrasound gel. Avoid bubbles in the contact region.</li>
		<li>Place the holder on the work stage for acoustic coupling. Perform steps 2.3&#8210;2.4 to start the laser and collect A-line signal. Perform step 2.6&#8210;2.7 to align. Press Stop to end collection after alignment.</li>
		<li>Select the RIM program. Name the newly created folder. Click the Collect button to start scanning.</li>
		<li>Click the Stop button to end scanning after completing acquisition. Click the Exit button to exit the RIM program.</li>
	</ol>
	</li>
	<li>Use the RIM data for reconstruction of the maximum amplitude projection (MAP) along the depth direction by user-defined program. Observe the dynamic changes in the animal.</li>
</ol>

<ol>
	<li>Li, L., 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=Single-impulse+panoramic+photoacoustic+computed+tomography+of+small-animal+whole-body+dynamics+at+high+spatiotemporal+resolution.">Single-impulse panoramic photoacoustic computed tomography of small-animal whole-body dynamics at high spatiotemporal resolution.</a> Nature Biomedical Engineering. 1 (5), 0071 (2017).</li><li>Jeon, S., Kim, J., Lee, D., Baik, J. W., 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=Review+on+practical+photoacoustic+microscopy.">Review on practical photoacoustic microscopy.</a> Photoacoustics. 15, 100141 (2019).</li><li>Baik, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super+wide-field+photoacoustic+microscopy+of+animal+and+humans+in+vivo.">Super wide-field photoacoustic microscopy of animal and humans in vivo.</a> IEEE Transactions on Medical Imaging. 39 (4), 975-984 (2019).</li><li>Omar, M., Aguirre, J., 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=Optoacoustic+mesoscopy+for+biomedicine.">Optoacoustic mesoscopy for biomedicine.</a> Nature Biomedical Engineering. 3 (5), 354-370 (2019).</li><li>Lin, L., 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=Single-breath-hold+photoacoustic+computed+tomography+of+the+breast.">Single-breath-hold photoacoustic computed tomography of the breast.</a> Nature Communications. 9 (1), 2352 (2018).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide-field+monitoring+and+real-time+local+recoding+microvascular+networks+on+small+animals+with+a+dual-raster-scanned+photoacoustic+microscope.">Wide-field monitoring and real-time local recoding microvascular networks on small animals with a dual-raster-scanned photoacoustic microscope.</a> Journal of Biophotonics. 13 (6), 202000022 (2020).</li><li>Sun, J., Zhou, Q., Yang, S. <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+imaging+guided+sclerotherapy+for+vascular+malformations:+a+feasibility+study.">Label-free photoacoustic imaging guided sclerotherapy for vascular malformations: a feasibility study.</a> Optics Express. 26 (4), 4967-4978 (2018).</li><li>Xu, D., Yang, S., Wang, Y., Gu, Y., Xing, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+and+high-resolving+photoacoustic+dermoscopy+of+human+skin.">Noninvasive and high-resolving photoacoustic dermoscopy of human skin.</a> Biomedical Optics Express. 7 (6), 2095-2102 (2016).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+photoacoustic+probe+for+in+vivo+imaging+of+subcutaneous+microvessels+within+human+skin.">Miniaturized photoacoustic probe for in vivo imaging of subcutaneous microvessels within human skin.</a> Quantitative Imaging in Medicine and Surgery. 9 (5), 807-814 (2019).</li><li>Chen, Q., 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=Ultracompact+high-resolution+photoacoustic+microscopy.">Ultracompact high-resolution photoacoustic microscopy.</a> Optics Letters. 43 (7), 1615-1618 (2018).</li><li>Lan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+widefield+photoacoustic+microscopy+of+small-animal+hemodynamics.">High-speed widefield photoacoustic microscopy of small-animal hemodynamics.</a> Biomedical Optics Express. 9, 4689-4700 (2018).</li><li>Ma, H., Yang, S., Cheng, Z., Xing, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+confocal+dermoscope+with+a+waterless+coupling+and+impedance+matching+opto-sono+probe.">Photoacoustic confocal dermoscope with a waterless coupling and impedance matching opto-sono probe.</a> Optics Letters. 42 (12), 2342-2345 (2017).</li><li>Kang, H., Lee, S. 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All animal experiments were performed according to the approved guidelines and regulations of the Institutional Animal Care and Use Committee. The authors have no relevant financial interests in the manuscript and no other potential conflicts of interest to disclose.

Here we presented a dual raster-scanning photoacoustic small-animal imager for noninvasive vascular visualization which was designed and developed to capture the structure of the vasculature and the related dynamic change of blood. The advantage of DRS-PAI is that it integrates the WIM and the RIM into one system, which makes it easier to study vascular dynamic and vascular network structure of small animals. The system can provide high-resolution wide-field vascular visualization and real-time blood dynamics.
<p cla...

In the basic biomedical field, small animals can simulate human physiological function. Therefore, small-animal imaging plays an important role in guiding the research of human homologous diseases and seeking effective treatment1. Photoacoustic imaging (PAI) is a non-invasive imaging technique combining the advantages of optical imaging and ultrasound imaging2. Photoacoustic microscopy (PAM) is a valuable imaging method for basic research of small animal3. PAM can easily obtain high-resolution, deep-penetration, high-specificity and high-contrast images based on optical excitation and ultrasound detection4.
A pulse laser with a specific wavelength is absorbed by endogenous chromophores of tissues. Subsequently, the temperature of the tissue rises, which results in the production of photo-induced ultrasonic waves. The ultrasonic waves can be detected by an ultrasonic transducer. After signal acquisition and image reconstruction, the spatial distribution of the absorber can be obtained5. On the one hand, the visualization of whole-organ vascular network requires a wide field of view. The process of wide-field scanning usually takes a long time to ensure high-resolution6,7,8. On the other hand, observing the hemodynamic activities of small animals requires fast real-time imaging. The real-time imaging is beneficial to study the vital signs of small animals in real time9,10,11. The field of view of real-time imaging is usually sufficiently small to ensure a high update rate. Thus, there is often a tradeoff between achieving a wide field of view and real-time imaging. Previously, two different systems were used for wide-field imaging or real-time imaging, separately.
This work reports a dual raster-scanning photoacoustic imager (DRS-PAI), which integrated wide-field imaging based on a two-dimensional motorized translation stage and real-time imaging based on a two-axis galvanometer scanner. The wide-field imaging mode (WIM) is performed to show vascular morphology. For the real-time imaging mode (RIM), there are currently two functions. First, RIM can provide real-time B-scan images. By measuring the displacement of vasculature along the depth direction, the characteristics of respiration or pulse can be revealed. Second, the RIM can quantitatively measure the specific area in the WIM image. By providing comparable images of local WIM regions, the details of the local change can be accurately revealed. The system designs a flexible transition between wide-field imaging of vascular visualization and real-time imaging of the local dynamic. The system is desirable in basic biomedical research where there is a need for small-animal imaging.

<table>
	<tbody>
		<tr>
			<td>12 bit multi-purpose digitizer</td>
			<td>Spectrum</td>
			<td>M3i.3221</td>
			<td>Data acquisition card</td>
		</tr>
		<tr>
			<td>A-line collected program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>RF Bay</td>
			<td>LNA-650</td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Depilatory Cream</td>
			<td>Veet</td>
			<td>33-II</td>
			<td>Animal depilatory</td>
		</tr>
		<tr>
			<td>Fiberport Coupler</td>
			<td>Thorlab</td>
			<td>PAF-X-7-A</td>
			<td>Fiber Coupler</td>
		</tr>
		<tr>
			<td>Field Programmable Gate Array</td>
			<td>Altera</td>
			<td>Cyclone IV</td>
			<td>Trigger Control</td>
		</tr>
		<tr>
			<td>Fixed Focus Collomation Packages</td>
			<td>Thorlabs</td>
			<td>F240FC-532</td>
			<td>Fiber Collimator</td>
		</tr>
		<tr>
			<td>Foused ultrasonic transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics Processing Unit</td>
			<td>NVIDIA</td>
			<td>GeForce GTX 1060</td>
			<td>Processing data</td>
		</tr>
		<tr>
			<td>Holder</td>
			<td>Self-made</td>
			<td></td>
			<td>Animal fixation</td>
		</tr>
		<tr>
			<td>Laser control program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Guangdong Medical Laboratory Animal Center</td>
			<td>BALB/c</td>
			<td>Animal Model</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Mshot</td>
			<td>MS60</td>
			<td>CCD camera</td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Daheng Optics</td>
			<td>GCO-2111</td>
			<td>Objective Lens</td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td>Daheng Optics</td>
			<td>GCC-1011</td>
			<td>Moveable/Fixed Mirror</td>
		</tr>
		<tr>
			<td>Moving Magnet Capacitive Detector Galvanometer Scanner</td>
			<td>Century Sunny</td>
			<td>S8107</td>
			<td>real-time scanner</td>
		</tr>
		<tr>
			<td>Mshot image analysis system</td>
			<td>Mshot</td>
			<td></td>
			<td>Display software</td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>CR DOUBLE-CRANE</td>
			<td>H34023609</td>
			<td>Normal Saline</td>
		</tr>
		<tr>
			<td>Ophthalmic Scissors</td>
			<td>SUJIE</td>
			<td></td>
			<td>Scalp Remove</td>
		</tr>
		<tr>
			<td>Planar ultrasonic transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Wrap</td>
			<td>HJSJLSL</td>
			<td></td>
			<td>Polyethylene Membrane</td>
		</tr>
		<tr>
			<td>Program Control Software</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined Program</td>
		</tr>
		<tr>
			<td>Pulsed Q-swithched Laser</td>
			<td>Laser-export</td>
			<td>DTL-314QT</td>
			<td>532-nm pulse Laser</td>
		</tr>
		<tr>
			<td>Real-time imaging program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Ring-shaped white LED</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Codos</td>
			<td>CP-9200</td>
			<td>Animal Shaver</td>
		</tr>
		<tr>
			<td>Single-Mode Fibers</td>
			<td>Nufern</td>
			<td>460-HP</td>
			<td>Single-mode fiber</td>
		</tr>
		<tr>
			<td>Surgical Blade</td>
			<td>SUJIE</td>
			<td>11</td>
			<td>Blade</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>SUJIE</td>
			<td>7</td>
			<td>Scalp Remove</td>
		</tr>
		<tr>
			<td>Translation Stage</td>
			<td>Jiancheng Optics</td>
			<td>LS2-25T</td>
			<td>wide-field scanning stage</td>
		</tr>
		<tr>
			<td>Ultrasonic Transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>GUANGGONG PAI</td>
			<td>ZC4252418</td>
			<td>Acoustic Coupling</td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Tokyo Chemical Industry</td>
			<td>C0028</td>
			<td>Animal Anestheized</td>
		</tr>
		<tr>
			<td>Water tank</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field imaging program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>XY Translator Mount</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dual raster-scanning photoacoustic small-animal imager for vascular visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

The schematic of the DRS-PAI is shown in Figure 1. The system allows flexible and repeatable switching between WIM with RIM. The acquired PA signal is processed quickly to generate PA B-Scan and MAP images. The CCD camera can provide photographs of samples.
All components of the DRS-PAI are integrated and assembled in an imager setup (Figure 2), making it easy to assemble and operate. In the WIM, continuous raster scanning of a two-di...

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Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

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4.0K Views.  South China Normal University. A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Video: Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Three-dimensional Optical-resolution Photoacoustic Microscopy

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

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

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

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

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 ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

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.

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.

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

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

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

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

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...