Name: switchable acoustic and optical resolution photoacoustic microscopy for in vivo small-animal blood vasculature imaging
Uploaded: 2017-06-26T15:00:00.000+00:00
Duration: 10 min 17 s
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 tunability range of 559-576 nm as the optical irradiation source. Set the laser wavelength to 570 nm using an external controller and the repetition rate of the laser to 1 kHz using the laser software.</li>
		<li>Place a beam sampler at a 45&#176; angle in front of the laser to divert 5% of the laser power to a photodiode through a variable neutral density filter (NDF1; OD = 0-4.0).</li>
		<li>Divert the laser beam after the beam sampler at 90&#176; using a right-angle prism (RAP1).</li>
		<li>Use another right-angle prism (RAP2) to allow the beam to pass through a variable neutral density filter (NDF2; OD = 0-4.0) and onto a multimode fiber (MMF), directing it through a fiber coupler (FC)-a combination of objectives (numerical aperture (NA): 0.25) and an XY translator.</li>
		<li>Fix the fiber onto the scanning stage using an XY translator. Place a plano-convex lens (L1) 25 mm away from the fiber output end to collimate the beam out of the fiber.</li>
		<li>Pass the collimated beam through a conical lens with an apex angle of 130&#176; to generate a ring-shaped beam. Weakly focus the ring-shaped beam onto the subject using a homemade optical condenser (OC) with cone angles of 70&#176; and 110&#176; and with a hole in the center.</li>
		<li>Place a 50 MHz ultrasonic transducer (UST) with an acoustic lens (AL) at the center of the homemade condenser.</li>
	</ol>
	</li>
	<li>System configuration: OR-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 tunability range of 559 - 576 nm as the optical irradiation source. Set the laser wavelength at 570 nm using an external controller and the repetition rate of the laser at 5 kHz using the laser software.</li>
		<li>Rotate the computer-controlled rotational stage (holding the RAP1) by 90&#176; to divert the laser beam onto an iris for reshaping.</li>
		<li>Attenuate the laser beam placing a variable neutral density filter (OD: 0-4.0) along the beam and then focus the beam with a condenser lens (CL). Pass it through a pinhole (PH) 75 mm away from the CL for spatial filtering.</li>
		<li>Launch the spatially filtered beam onto a single-mode fiber (SMF) using a single-mode fiber coupler (FC) consisting of a 0.1 NA objective to focus the light beam on to the SMF.</li>
		<li>Adjust the fiber coupler to achieve maximum coupling efficiency.</li>
		<li>Fix the fiber out onto the scanning stage using a slip plate (SP). Place an achromatic lens (L2) 50 mm away from the SM fiber to collimate the laser beam.</li>
		<li>Divert the collimated beam by 90&#176; using a kinematic controllable elliptical mirror (M) to fill the back aperture of another identical achromatic lens (L3). Place the achromatic lens used for focusing on a translation mount (TM2) using a lens tube (LT).</li>
		<li>Pass the focusing beam through a homemade optoacoustic beam combiner consisting of a right-angled prism (RA) and a rhomboid prism (RP), with a layer of silicon oil (SO) in between. 
		NOTE: The silicon oil layer will act as optically transparent and acoustically reflective film.</li>
		<li>Attach an acoustic lens (AL) to provide acoustic focusing (focal diameter: ~46 &#181;m) at the bottom of the rhomboid prism.</li>
		<li>Place the ultrasonic transducer with a 50 MHz center frequency on top of the rhomboid prism; use an epoxy layer for effective coupling.</li>
	</ol>
	</li>
</ol>
2. System Switching and Alignment
<ol>
	<li>Fix (by screwing tightly) the homemade switchable plate to a 3-axis motorized stage controlled by a 3-axis controller connected to the computer.</li>
	<li>Attach the AR and OR cage system to the homemade plate using cage mounting brackets to allow for easy switching between the AR and OR scan heads. Slide the scan head on top of the imaging area.</li>
	<li>Use the Z-stage to submerge the bottom part of the AR-OR-PAM scanner head in a water-filled acrylic tank (13 cm x 30 cm x 3 cm) for acoustic coupling.</li>
	<li>Open an imaging window with a 7-cm diameter on the bottom plate of the tank and seal it with a polyethylene membrane for optical and acoustic transmission.</li>
	<li>Use a pulse-echo amplifier and an oscilloscope to align the ultrasound transducer in focus.
	<ol>
		<li>Set the gain in pulse echo amplifier to 24 db in the transmission/receiving mode.</li>
		<li>Use the sync-out signal from the pulse-echo amplifier as the trigger and detect the backscattered signal from a glass slide (inserted from the bottom of water tank) using an oscilloscope. 
		NOTE: The slide should have black tape stuck to it.</li>
		<li>Move the Z-axis to maximize the amplitude of the pulse-echo signal (viewed on the oscilloscope). 
		NOTE: When the glass plate is in focus, the echo will have its maximum amplitude.</li>
	</ol>
	</li>
	<li>Switch on the laser and connect the UST to two amplifiers, each with 24 dB fixed gain, using BNC cables. 
	NOTE: The outputs of the amplifiers are connected to the data acquisition card (DAQ).</li>
	<li>Use the signal from the photodiode (PD) placed in front of the laser as a trigger for the data acquisition system.</li>
	<li>In AR-PAM, vary the distance between the conical lens (con.L) and the optical condenser (OC) to maximize the amplitude of the photoacoustic signal generated from the test object (black tape stuck on a glass slide). Ensure that the optical and acoustic focuses are confocal by determining the maximum photoacoustic (PA) signal amplitude.
	<ol>
		<li>Note the delay of the maximum PA signals; use this later to check the focus in the data acquisition software.</li>
	</ol>
	</li>
	<li>Loosen the screw of scanning head and manually switch the scanning head from AR-PAM to OR-PAM. Then, tighten the screws.</li>
	<li>In OR-PAM, vary the distance between the focusing achromatic doublet (inside the lens tube (LT)) and the optoacoustic combiner to maximize the PA signal amplitude shown on the oscilloscope.
	<ol>
		<li>Note the delay of the maximum PA signals. 
		NOTE: Finetuning is necessary to determine the confocal arrangement.</li>
	</ol>
	</li>
</ol>
3. Experimental Steps
<ol>
	<li>Lateral resolution and imaging depth quantification
	<ol>
		<li>Use gold nanoparticles 100 nm in diameter to determine the lateral resolution of the AR and OR system.</li>
		<li>Dilute 0.1 mL of nanoparticle solution with an equal amount of water. Distribute 0.1 mL of diluted solution on a cover slip and place it in contact with the polyethylene membrane under the tank.</li>
		<li>Make sure that the AR-PAM and OR-PAM is in focus in the data acquisition software (see the Table of Materials) before scanning (steps 2.8 and 2.10). 
		NOTE: By knowing the microsecond delay of the maximum PA signals from steps 2.9 and 2.10, multiplied by the sampling rate (250 MS/s), the image will be in focus in the data acquisition software. The delay which that must be omitted during data acquisition can be determined in the software so as to save only the necessary data points for post-processing.</li>
		<li>Set the scan parameters for the AR-PAM and press the &#34;scan&#34; button to start raster scanning.
		<ol>
			<li>Set the scan parameters for the AR-PAM in the data acquisition software at &#34;4&#34; mm/s scanning speed in the &#34;velocity&#34; tab, &#34;1&#34; kHz in the &#34;pulse repetition rate&#34; tab, &#34;0.5&#34; mm in the &#34;Y-scan range&#34; tab, and &#34;0.5&#34; mm in the &#34;X-scan range&#34; tab. Set the step size in the x-direction at &#34;4&#34; &#181;m in the &#34;dx&#34; tab. 
			NOTE: The step size in the y-direction is automatically determined from the scan speed velocity of the stage and the pulse repetition rate (in this case, 4,000 &#181;m/1,000 Hz = 4 &#181;m)</li>
		</ol>
		</li>
		<li>Set the scan parameters for the OR-PAM and press the &#34;scan&#34; button to start raster scanning.
		<ol>
			<li>Set the scan parameters in the data acquisition software at &#34;2.5&#34; mm/s scanning speed in the &#34;velocity&#34; tab, &#34;5&#34; kHz in the &#34;pulse repetition rate&#34; tab, &#34;0.5&#34; mm in the &#34;Y-scan range&#34; tab, and &#34;0.5&#34; mm in the &#34;X-scan range&#34; tab. Set the step size in the x-direction a &#34;0.5&#34; &#181;m in the &#34;dx&#34; tab. 
			NOTE: The step size in the y-direction is automatically determined from the scan speed velocity of the stage and the pulse repetition rate (in this case, 2,500 &#181;m/5,000 Hz = 0.5 &#181;m).</li>
		</ol>
		</li>
		<li>Ensure that during the scanning process, the data is continuously captured and stored on the computer 
		NOTE: Data will be captured only in one direction of motion of the Y-stage.</li>
		<li>Use the multiple B-scan data stored in the computer to retrieve the maximum amplitude projection (MAP) images using image processing software (see the Table of Materials).</li>
		<li>Use a single nanoparticle image (out of multiple images) from the scan to determine the lateral resolution by manually plotting a line through the central region of the nanoparticle image to obtain a point-spread function, which looks like Gaussian curve. See Figure 2.</li>
		<li>Fit the point-spread function obtained from a single nanoparticle image using a Gaussian fit function and measure the full width at half maximum (FWHM) using image processing software (see the Table of Materials). Use this as the lateral resolution. See Figure 2.</li>
		<li>Insert a piece of black tape obliquely onto a piece of sliced chicken tissue as the target object for depth imaging. Place the tissue with the tape in the water tank. 
		NOTE: The black tape is stuck to a metal plate with a sharp tip, which helps to attach the tape to the tissue.</li>
		<li>Set the scan parameters for the AR-PAM into the data acquisition software and then press the &#34;scan&#34; button to capture a single B-scan image to determine the maximum imaging depth.
		<ol>
			<li>Set the scan parameters at &#34;15&#34; mm/s scanning speed in the &#34;velocity&#34; tab, &#34;1&#34; kHz in the &#34;pulse repetition rate&#34; tab, &#34;5&#34; cm in the &#34;Y-scan range&#34; tab, and &#34;0.1&#34; mm in the &#34;X-scan range&#34; tab. Set the step size in the x-direction at &#34;0.1&#34; mm in the &#34;dx&#34; tab.</li>
		</ol>
		</li>
		<li>Set the scan parameters for the OR-PAM and press the &#34;scan&#34; button to capture a single B-scan image to determine the maximum imaging dept.
		<ol>
			<li>Set the scan parameters in the data acquisition software as &#34;15&#34; mm/s scanning speed in the &#34;velocity&#34; tab, &#34;5&#34; kHz in the &#34;pulse repetition rate&#34; tab, &#34;2&#34; cm in the &#34;Y-scan range&#34; tab and &#34;0.1&#34; mm in the &#34;X-scan range&#34; tab. Set the step size in the x-direction at &#34;0.1&#34; mm in the &#34;dx&#34; tab. 
			NOTE: Since the X-scan range and dx are the same, only one B-scan will be captured. The time-resolved PA signals multiplied by the speed of sound in soft tissue (1,540 m/s) will give an A-line image. Multiple A-lines are captured during the continuous motion of the Y-stage to produce a B-scan.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>In vivo imaging of the mouse ear blood vasculature
	<ol>
		<li>Use a female mouse with a bodyweight of 25 g and an age of 4 weeks.</li>
		<li>Anesthetize the animal using a cocktail of ketamine (120 mg/kg) and xylazine (16 mg/kg) injected intraperitoneally (dosage of 0.1 mL/10 g).</li>
		<li>Remove the hair from the animal ear using hair removal cream. Wipe the area clean. Cover the eye of the animal with a sterile ocular ointment to avoid any scattered laser beam falling onto the eyes.</li>
		<li>Position the animal on a stage that also has a miniature plate to position the ear.</li>
		<li>Maintain anesthesia with inhaled isoflurane (0.75% in 1 L/min oxygen) during the imaging period.</li>
		<li>Clamp a pulse oximeter to the mouse leg or tail and monitor the physiological status. Allow the imaging region to be in contact with the polyethylene membrane using ultrasound gel.</li>
		<li>Set the scan parameters for the AR-PAM and press the &#34;scan&#34; button to start raster scanning.
		<ol>
			<li>Set the scan parameters for the AR-PAM in the data acquisition software at &#34;15&#34; mm/s scanning speed in the &#34;velocity&#34; tab, &#34;1&#34; kHz in the &#34;pulse repetition rate&#34; tab, &#34;10 mm&#34; in the &#34;Y-scan range&#34; tab, and &#34;6&#34; mm in the &#34;X-scan range&#34; tab. Set the step size in the x-direction as &#34;30&#34; &#181;m in the &#34;dx&#34; tab. 
			NOTE: The step size in the y-direction is automatically determined from the scan speed velocity of the stage and the pulse repetition rate (in this case, 15,000 &#181;m/1,000 Hz = 15 &#181;m).</li>
		</ol>
		</li>
		<li>After finishing AR-PAM scan, switch the imaging head position from AR-PAM to OR-PAM (as described in section 2).</li>
		<li>Set the scan parameters for the OR-PAM and press the &#34;scan&#34; button to start raster scanning.
		<ol>
			<li>Set the scan parameters for the OR-PAM in the data acquisition software at &#34;15&#34; mm/s scan speed in the &#34;velocity&#34; tab, &#34;5&#34; kHz in the &#34;pulse repetition rate&#34; tab, &#34;10&#34; mm in the &#34;Y-scan range&#34; tab, and &#34;6&#34; mm in the &#34;X-scan range&#34; tab. Set the step size in the x-direction as &#34;6&#34; &#181;m in the &#34;dx&#34; tab. 
			NOTE: The step size in the y-direction is automatically determined from the scan speed velocity of the stage and the pulse repetition rate (in this case, 15,000 &#181;m/5,000 Hz = 2 &#181;m).</li>
		</ol>
		</li>
		<li>Use the multiple B-scan data stored in the computer to retrieve the MAP images using image processing software.</li>
		<li>Observe the animal during the entire imaging period.</li>
	</ol>
	</li>
</ol>

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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>

All animal experiments were performed according to the approved guidelines and regulations of the Institutional Animal Care and Use Committee of Nanyang Technological University, Singapore (Animal Protocol Number ARF-SBS/NIE-A0263). The authors have no relevant financial interests in the manuscript and no other potential conflicts of interest to disclose.

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

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Research

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Bioengineering

11.8K Views.  Nanyang Technological University. 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.

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

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 ...

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 ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

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

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. ...

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.

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 ...

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 ...

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

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Three-dimensional Optical-resolution Photoacoustic Microscopy

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

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

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

Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research