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 border="0" cellpadding="0" cellspacing="0" width="939">
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Q-switched Nd:YAG laser</td>
			<td style="width:232px;">Edgewave</td>
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			<td height="21" style="height:21px;">Precision Linear Stage</td>
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			<td>VT 80&#160;</td>
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			<td height="21" style="height:21px;">Mounted Silicon photodiode</td>
			<td>Thorlabs</td>
			<td>SM05PD1A</td>
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			<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>
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			<td height="21" style="height:21px;">Mounted Continuously Variable ND Filter</td>
			<td>Thorlabs</td>
			<td>NDC-50C-4M</td>
			<td>Intensity variable</td>
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			<td height="21" style="height:21px;">Fiber Patch Cable</td>
			<td>Thorlabs</td>
			<td>M29L01</td>
			<td>Multimode fiber</td>
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		<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>
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			<td height="21" style="height:21px;">XY translating mount</td>
			<td>Thorlabs</td>
			<td>CXY1</td>
			<td>Translating mount</td>
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			<td height="21" style="height:21px;width:303px;">Plano convex lens</td>
			<td>Thorlabs</td>
			<td>LA1951</td>
			<td>Collimating lens</td>
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		<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>
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		<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>
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			<td height="21" style="height:21px;">Pulser/Receiver</td>
			<td>Olympus-NDT</td>
			<td>5073PR</td>
			<td>Pulse echo amplifier&#160;</td>
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			<td height="21" style="height:21px;width:303px;">Mounted standard iris</td>
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			<td>ID12/M</td>
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			<td height="21" style="height:21px;width:303px;">Plano convex lens</td>
			<td>Thorlabs</td>
			<td>LA4327</td>
			<td>Condenser lens</td>
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			<td height="21" style="height:21px;width:303px;">Mounted precision pinhole</td>
			<td>Thorlabs</td>
			<td>P50S</td>
			<td>Spatial filtering</td>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Lens tube</td>
			<td>Thorlabs</td>
			<td>SM05L10</td>
			<td></td>
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		<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>
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		<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>
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		<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 ...

switchable-acoustic-optical-resolution-photoacoustic-microscopy-for

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JoVE Journal

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 ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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 ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

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, ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...