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>

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	<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...

Watch this Scientific Journal Video about Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization at JoVE.com

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 ...

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

Research

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Bioengineering

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 ...

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 ...

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.

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 ...

Engineering

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Neuroscience

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 ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. Custom-made circular ring array-based transducers and conventional bulky Nd:YAG/OPO lasers inhibit translation of the PAT system to clinics. Ultra-compact pulsed laser diodes (PLDs) are currently being used as an alternative source of near-infrared excitation for PA imaging. High-speed dynamic in vivo imaging has been demonstrated using a compact PLD-based desktop PAT system (PLD-PAT). A visualized experimental protocol using the desktop PLD-PAT system is provided in this work for dynamic in vivo brain imaging. The protocol describes the desktop PLD-PAT system configuration, preparation of animal for brain vascular imaging, and procedure for dynamic visualization of indocyanine green (ICG) dye uptake and clearance process in rat cortical vasculature.&#160;

A compact pulsed laser diode-based desktop photoacoustic tomography (PLD-PAT) system is demonstrated for high-speed dynamic in vivo imaging of small animal cortical vasculature.

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. ...

Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the development of live imaging systems has set the (analytical) pace in both basic and clinical research, with the objective of creating new instruments capable of providing real-time, quantifiable endpoints with clinical interest and application. Near-infrared spectroscopy (NIRS), positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) are available, among other techniques, but cost, image resolution, and reduced contrast are recognized as common challenges. Optoacoustic tomography (OT) offers a new perspective on vascular functional imaging, combining state-of-the-art optical absorption and spatial resolution capacities (from micrometer optical to millimeter acoustic resolution) with tissue depth. In this study, we tested the applicability of multispectral optoacoustic tomography (MSOT) for functional imaging. The system uses a tunable optical parametric oscillator (OPO) pumped by an Nd: YAG laser, providing excitation pulses sensed by a 3D probe at wavelengths from 680 nm to 980 nm. Images obtained from the human forearm were reconstructed through a specific algorithm (supplied within the manufacturer&#39;s software) based on the response of specific chromophores. Maximal Oxygenated Hemoglobin (Max HbO2) and Deoxygenated Hemoglobin (Max Hb), Total Hemoglobin (HbT), and mean Oxygen Saturation (mSO2) to vascular density (&#181;Vu), inter-unit average distances (&#950;Ad), and capillary blood volume (mm3) may be measured using this system. The applicability potential found with this OT system is relevant. Ongoing software developments will surely improve the utility of this imaging system.

The present protocol describes the acquisition of multispectral optoacoustic images of in vivo human skin vasculature. These include the quantification of hemoglobin and melanin, regarded as chromophores of interest for functional analysis.

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the ...

Non-Invasive Integrated PAUSAT Whole-Brain Imaging of Ischemic Stroke

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.

Author Spotlight: Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke  

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging ...

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

Summary

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Chapters in this video

Three-dimensional Optical-resolution Photoacoustic Microscopy

Photoacoustic Cystography

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

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

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

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

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke