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

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

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

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

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

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

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

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

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

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

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

The overall goal of this procedure is to demonstrate a switchable acoustic and optical resolution photoacoustic microscopy system for in vivo small-animal blood vasculature imaging. Photoacoustic microscopy is a fast-growing in vivo imaging modality that combines optics and ultrasound, providing imaging depth beyond optical path with high resolution. This work present is switchable acoustic optical resolution photo acoustic microscopic system capable of higher-resolution imaging at shallow depth as well as lower-resolution deep tissue imaging of the same sample in vivo.Demonstrating the procedure will be Dr.Mohesh Moothanchery, a research fellow from my laboratory, and Arunima Sharma, a PhD student from my lab. First, construct a nanosecond, tunable laser system from a diode-pumped, solid-state Nd:YAG laser and a ditunable laser with a range of 559 to 576 nanometers. Set the ditunable laser to 570 nan.Mount on an optical table an acrylic tank with a seven-centimeter polyethylene imaging window in the floor of the tank. Fix an imaging stage to an optical post beneath the imaging window. Over the tank, mount an optical cage containing the AR-OR-PAM imaging system on a switching plate fastened to a computer-controlled three-axis motorized translation stage.To configure the system for AR-PAM, use a right angel prism mounted on a motorized continuous rotational stage to direct the beam through a variable neutral-density filter to a multi-mode fiber cable with a 25 NA fiber coupler. Connect the fiber output to an XY translation stage in the imaging system. Direct the beam through a plano convex lens 25 millimeters from the output, and then through a conical lens to create a ring shaped beam.Focus the ring shaped beam on an optical condenser around a 50 megahertz ultrasonic transducer with an acoustic lens mounted at the transducer output. Next, to configure the system for OR-PAM, rotate the first right angle prism by 90 degrees to direct the laser beam through an iris, a variable neutral-density filter, a condenser lens, and a pinhole set at 75 millimeters from the condenser lens. Use a 1 NA single-mode fiber coupler and a single-mode fiber to send the beam to the Z translation stage in the imaging system.Direct the beam through an achromatic doublet lens mounted 50 millimeters from the fiber output. Divert the beam with a kinematic, controllable, elliptical mirror to a second achromatic doublet lens in a lens tube. Focus the beam on a right-angle prism separated by a layer of silicon oil from a rhomboid prism, bearing an acoustic lens and a 50 megahertz ultrasonic transducer, which forms the OR-PAM scanning head.Prior to beginning the system alignment, fill the acrylic tanks with degassed water. Then use the three-axis motorized stage to move the scanning assembly over the tank. Lower the assembly until the acoustic lenses of both systems are submerged.Turn on the laser. Connect the ultrasound transducers to two 25-decibel, fixed-gain amplifiers. Position the AR-PAM scanning head over the imaging window.Place a glass slide wrapped in black electrical tape on the imaging stage and raise the stage to bring the slide into contact with the imaging window. Adjust the conical lens until the photoacoustic signal amplitude generated from the slide reaches a maximum, indicating that the optical and acoustic lenses are confocal. Then manually switch to the OR-PAM system.Adjust the achromatic doublet lens until the optical and acoustic lenses are confocal, as indicated by maximizing the photoacoustic signal amplitude from the tape-covered slide. To determine the lateral resolution of each system, first place 1 milliliter of a dilute solution of 100 nanometer gold nanoparticles in water on a a cover slip. Place the slide on the imaging stage.and raise the stage until the nanoparticle solution contacts the imaging window. Switch the laser and the scanning head to the AR-PAM system. Configure the instrument software for an AR-PAM scan and perform a single raster scan.Repeat this process for the OR-PAM system. Then remove the slide and clean the imaging window with an alcohol swab. Fit the point spread functions determined from the acquired images to a Gaussian curve.The full width at half maximum is the lateral resolution for the corresponding scanning system. Next, to determine the maximum imaging depth in tissue, first insert a sharp metal plate wrapped in black tape into a small section of chicken tissue at a shallow angle. Place the tissue in the water tank under the scanner head.Obtain a single B-scan image for each system. Measure the depth beneath the tissue surface at which the black tape is no longer clearly discernible. Prior to image, ensure that the polyethylene imaging window and the animal imaging stage are both clean.Then obtain a 25-gram, four-week-old female mouse for the imaging procedure after anesthetizing the mouse. Remove hair from the ear surface with depilatory cream. Apply sterile ocular ointment to the mouse's eyes to prevent dryness and block scattered laser beams.Place the mouse on an imaging stage with the plate for positioning the ear to me imaged. Monitor the mouse's physiological condition with a pulse oximeter clamped to the mouses tail or leg. Apply ultrasound gel to the ear to be imaged.Slowly raise the imaging stage to gently bring the ear into contact with the polyethylene imaging window. Acquire AR-PAM and OR-PAM raster scans of the mouse's ear, monitoring the mouse's physiological status throughout. Allow the mouse to recover fully after imaging.The blood vasculature system of a mouse here was imaged in vivo with a switchable AR-OR-PAM system. Blood vessels thicker than 45 micrometers were clearly visible in the AR-PAM image. Single capillaries of about five micrometers wide were resolved with OR-PAM imaging.The combined AR-OR-PAM system has an approximately four micron lateral resolution. And at point, four mm imaging for the OR-PAM and an approximately 45 micrometer lateral resolution and 7.8 mm imaging for the AR-PAM. The switchable combined AR-OR-PAM system allows imaging without moving the sample between different imaging systems.The developed system can be used for pre-clinical imaging. Major pre-clinical applications include imaging of androgenesis, tumor micro-environments, microcirculations, drug response, brain function, bio-markers, and gene activities.

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11.8K Views.  Nanyang Technological University. The overall goal of this procedure is to demonstrate a switchable acoustic and optical resolution photoacoustic microscopy system for in vivo small-animal blood vasculature imaging. Photoacoustic microscopy is a fast-growing in vivo imaging modality that combines optics and ultrasound, providing imaging depth beyond optical path with high resolution. This work present is switchable acoustic optical resolution photo acoustic microscopic system capable of higher-resolution imaging at shallow de...