The authors would like to acknowledge the financial support from the Hong Kong Innovation and Technology Commission (ITS/036/19).

All animal experiments performed in this work are approved by the Animal Ethics Committee at The Hong Kong University of Science and Technology.
1. Open-top UV-PAM system (Figure 1)
<ol>
	<li>Optical illumination
	<ol>
		<li>Use a Q-switch diode-pumped solid-state laser (266 nm wavelength) as an excitation source.</li>
		<li>Use two convex lenses to expand the laser beam and place a pinhole close to the focal point of the first convex lens to perform spatial filtering.</li>
		<li>Reflect the laser beam upward using a 1D galvanometer mirror (1D GM).</li>
		<li>Use an objective lens to focus the laser beam and pass through the center of a ring-shaped ultrasonic transducer (UT) before being tightly focused on a sample.</li>
	</ol>
	</li>
	<li>Ultrasonic detection
	<ol>
		<li>Use a ring-shaped focused UT to detect ultrasonic waves. The inner and outer diameters of the UT active area are 3 mm and 6 mm, respectively. Focal length: 6.3 mm; center frequency: 40 MHz; -6 dB bandwidth: 84%.</li>
		<li>Fix the UT into a lab-made water tank with an optically transparent window at the bottom, covered by a thin quartz coverslip to allow UV light to pass through. Ensure that the active area of the UT is facing upward. Attach the water tank to a two-axis manual stage for controlling the lateral position of the UT.</li>
		<li>Turn on the UV laser, adjust the position of the UT to allow the laser beam to pass through from the center of the UT. Turn off the UV laser. Then, fill the water tank with deionized water to fully immerse the UT.</li>
		<li>Connect the output of the UT to two amplifiers (total gain = 56 dB) and connect the output of the second amplifier to a data acquisition card (DAQ).</li>
	</ol>
	</li>
	<li>Attach a sample holder to a z-axis manual stage which is connected to xy-motorized stages. The sample holder has an empty hole that allows UV light to pass through. Attach four pieces of double-sided tape surrounding the hole.</li>
	<li>System alignment
	<ol>
		<li>Attach black tape to a glass slide and place the glass slide to cover the hole of the sample holder, with the black tape facing downward. Press the glass slide to ensure that it is fixed on the sample holder. Then, lower the sample holder to immerse the glass slide into the water.</li>
		<li>Disconnect the ring-shaped UT and amplifiers, connect the UT to the pulser/receiver, and connect the output of the pulser/receiver to an oscilloscope. Operate the pulser/receiver into the Pulse-Echo mode. Set the pulse amplitude and the gain of the pulser/receiver to 6 and 20 dB, respectively.
		<ol>
			<li>Enable the pulser/receiver and adjust the z-position of the sample holder to find the position of the acoustic focal plane where the ultrasonic signals are maximum.</li>
		</ol>
		</li>
		<li>Change the pulser/receiver to the transmission mode and set the gain to 60 dB. Enable the laser output. Adjust the z-position of the objective lens to maximize the PA signals that are measured by the oscilloscope.</li>
		<li>Adjust the lateral position of the ring-shaped UT to make the generated PA signal symmetric and maximum, which represents that the optical and acoustic foci are coaligned in the lateral plane. Then, adjust the z-position of the sample holder to maximize the PA signals.</li>
		<li>Repeat steps 1.4.3 and 1.4.4 to optimize both the symmetry and amplitude of the PA signals. Record the time delay (i.e., the time it takes for the PA waves to reach the UT) on the oscilloscope when the PA signals are optimized.</li>
		<li>Move the sample holder to image different positions of the black tape. Adjust the sample holder&#39;s flatness such that the PA signals generated from each position of the black tape have the same time delay as the one measured in step 1.4.5.</li>
		<li>Turn off the laser and connect the UT to the two amplifiers.</li>
	</ol>
	</li>
</ol>
2. Sample preparation
<ol>
	<li>FFPE mouse brain slice
	<ol>
		<li>Sacrifice a mouse with an overdose of anesthesia. Then, harvest the mouse brain following the protocol described in reference18.</li>
		<li>Fix the harvested brain in 10% neutral buffered formalin for 24 h.</li>
		<li>Process the fixed brain by dehydration with graded alcohol, clearing with xylene, and embedding with paraffin as described in reference19. 
		NOTE: Process the sample in a fume hood.</li>
		<li>Slice the embedded brain in thin slices (5 &#181;m in thickness) using a microtome. Place the sample slices on quartz slides. Dry the slides in an oven at 60 &#176;C for 1 h.</li>
		<li>Deparaffinize the sections using a clearing agent (see Table of Materials), which removes the paraffin to avoid high background signals as paraffin is highly absorbing with UV excitation. 
		NOTE: Process the sample in a fume hood.</li>
	</ol>
	</li>
	<li>Fresh mouse brain slice
	<ol>
		<li>Sacrifice a mouse with an overdose of anesthesia. Then, harvest the mouse brain following the protocol stated in reference18.</li>
		<li>Wash the mouse brain with phosphate-buffered saline (PBS) to remove the blood.</li>
		<li>Cut a slice of the brain sample (~5 mm thick) by hand, and then wash the sample with PBS to remove the blood on the cross section.</li>
	</ol>
	</li>
</ol>
3. Experimental procedures
<ol>
	<li>Sample placement
	<ol>
		<li>Prepare a lab-made sample tank with a UV transparent membrane (polyethylene, &#8764;10 &#181;m in thickness). Add a drop of water to the membrane and then place the biological sample on the sample tank to cover the water. In the case of the FFPE slice sample, use tape to fix the glass slide on the membrane.</li>
		<li>Place the sample-containing sample tank on the sample holder, ensuring to cover the empty hole of the sample holder.</li>
	</ol>
	</li>
	<li>Set the UV laser to the external-trigger mode. Using our lab-built LabVIEW program (see Table of Materials), set the scanning parameters as follows.
	<ol>
		<li>Set the laser repetition rate to 55 kHz; set the signal type of the GM driver to triangular and the driving voltage range to 0.018 V (representing &#177;0.018 V; scale factor: 1 V/&#176;). Set GMnum to 22 and dy to 2 so that after every 22 laser triggers, the GM finishes a quarter of the scanning period, and the y-axis motor moves a step size of 0.3125 &#181;m. Set dx to 192 so that when the y-axis motor reaches the preset position (e.g., 5 mm), the x-axis motor moves a step size of 30 &#181;m. 
		NOTE: The smallest incremental step size is set to be 0.15625 &#181;m for both the x- and y-axis motors. Therefore, when dy = 2, step size equals 0.15625 x 2 = 0.3125 &#181;m, and when dx = 192, step size equals 0.15625 x 192 = 30 &#181;m. To scan the entire area of 5 x 5 mm2, the x-axis motor will move 5000 &#181;m /30 &#181;m = 167 times, which means that 167 sub-images would be stitched to obtain a whole image. See Figure 2 for the scanning trajectory of the UV-PAM system.</li>
	</ol>
	</li>
	<li>Start trial scanning on a small region by setting the number of moving steps of the x- and y-axis motors (xn = 10 and yn = 1000). Adjust the z-axis manual stage to place the sample on the focal plane for obtaining maximum PA signals.</li>
	<li>Move both x- and y-axis motors to the desired starting point, set the scanning region by setting the xn and yn values, and then start the image acquisition program (see Table of Materials).</li>
	<li>After the image acquisition, turn off the laser, remove the sample holder, and store the biological samples.</li>
	<li>For fresh biological tissues, store the samples in 10% neutral buffered formalin.</li>
	<li>For the FFPE mouse brain slices, stain with H&#38;E following the protocol stated in reference20 to obtain the corresponding H&#38;E-stained images.</li>
	<li>Use the collected PA signals to reconstruct the maximum amplitude projection image using a lab-built image processing algorithm (see Table of Materials).</li>
</ol>

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T. T. W. W. and V. T. C. T. have a financial interest in PhoMedics Limited, which, however, did not support this work. The authors declare no conflicts of interest.

In summary, a high-speed and open-top UV-PAM system has been demonstrated for histological imaging. The detailed instructions about the system configuration, optical alignment, sample preparation, and experimental procedures are presented. The image acquisition program can be accessed from Github via the link provided in the Table of Materials. The lateral resolution of the present system is ~1.2 &#181;m which has been experimentally measured in a recent publication21. A mouse brain slice was imaged to demonstrate that the present system can obtain a histological image within 18 min for an area of 5 x 5 mm2, which is a typical size of brain biopsy22. Although the original image is in grayscale, with the assistance of a digital virtual staining tool enabled by a deep-learning algorithm, the present system can further provide virtually stained images in near real-time, ensuring easy adaptation for pathologists to interpret the images. As a label-free image technique, the present UV-PAM system can also provide histological images for unprocessed fresh tissue samples. More examples (including frozen-sectioned and fresh tissue samples) have been demonstrated in a previous publication17. The experimental results show the high potential of the present deep-learning-assisted UV-PAM system in SMA applications.
One of the advantages of the UV-PAM system is that the system is implemented in reflection mode, enabling imaging of thick tissues. Besides, the open-top UV-PAM system allows the sample to be placed on the scanning window (the membrane of the sample tank), which has a similar operation as traditional optical microscopies. Therefore, this system is more user-friendly than other systems that require samples to be sandwiched by two membranes11,14. Moreover, by using a 1D GM with a high-repetition-rate UV laser, the present UV-PAM system can achieve high imaging speed with higher cost-effectiveness when compared with the system using multifocal excitation23.
Currently, the imaging speed is mainly limited by the repetition rate of the laser and photon budget. With a laser that has a high repetition rate and high pulse energy, the imaging time can be further shortened. Another limitation of the system is that the sample can only be roughly adjusted to the focal plane of the objective lens by finding the maximum PA signals, instead of displaying a real-time image for users to visualize whether the sample is in focus. To display a near real-time image, 2D GM can be applied.
There are two critical steps in the protocol: (a) the confocal requirement of the optical and acoustic foci should be optimized to achieve a high detection sensitivity; (b) the scanning range of the GM on the x-axis should be smaller than the acoustic focal spot of the ring-shaped UT to maintain similar high detection sensitivity (in the current setup, the scanning range is ~30 &#181;m &#177;15 &#181;m). Otherwise, obvious vignetting effects around the edges would occur when multiple sub-images are stitched together to obtain a whole image.

Surgical margin analysis (SMA), which requires an examination of tissue specimens under a microscope, is an essential procedure to determine whether all cancer cells are removed from a patient's body in a resection surgery1. Therefore, a microscope that can rapidly provide histological images is vitally important for SMA to avoid repeated surgeries caused by incomplete removal of cancer cells. However, according to the current gold-standard method based on bright-field optical microscopy, the excised tissue is required to be fixed in formalin, embedded in paraffin, sectioned into thin slices (4-7 &#181;m), and then stained by hematoxylin and eosin (H&amp;E) before imaging, which is time-consuming (3-7 days) and laborious2,3. A frozen section is a rapid alternative for SMA by quickly freezing, slicing, and staining the tissue, which can provide histological images in 20-30 min4. However, the histological features are often distorted and required skillful training, which hinders the applicability of the technique to multiple types of organs5.
Optical microscopy techniques that can provide cellular images without or with a few steps of tissue processing have been developed for SMA. However, each of them suffers from different issues. For example, optical coherence tomography6 and confocal reflectance microscopy7 suffer from low specificity because of their low intrinsic scattering contrast. Although microscopy with ultraviolet surface excitation8 and light-sheet microscopy9 can provide high-resolution and high-contrast images for SMA, the toxic and volatile staining procedure usually cannot be performed in an operating room, which prolongs the turnaround time. Multi-photon microscopy10 and stimulated Raman microscopy11 can provide rich information for SMA. Yet, the high cost of the required ultrafast lasers that are used to generate nonlinear effects prevents their wide applicability.
Recently, by taking advantage of intrinsic optical absorption, label-free ultraviolet photoacoustic microscopy (UV-PAM) has been developed to provide high-resolution histological images12. In UV-PAM, the photon energy of the excitation UV light (e.g., 266 nm) is first absorbed by the DNA/RNA in cell nuclei13 and then converted into heat, inducing acoustic wave emission through thermal-elastic expansion14. By detecting the generated acoustic waves, two-dimensional (2D) UV-PAM images of cell nuclei can be obtained via maximum amplitude projection of the acoustic signals, providing histological information for SMA. To enable the clinical applications of UV-PAM, high-speed UV-PAM based on galvanometer mirror scanning has been developed to provide histological images for a brain biopsy sample (5 mm x 5 mm) within 18 min, showing great potential in time-sensitive applications15. To further validate the possibility of UV-PAM for thick tissue imaging, a reflection-mode UV-PAM system with a waterproof one-axis microelectromechanical systems scanner was proposed, successfully demonstrating intraoperative histopathological examination of human colon and liver tissues16. Since the original UV-PAM image is in grayscale while the gold standard H&amp;E-stained image is in pink and purple colors, it is difficult for pathologists to interpret UV-PAM images directly. To address this issue, a deep-learning algorithm was proposed to transfer grayscale UV-PAM images into virtual H&amp;E-stained images in near real-time so that pathologists can understand the images without any additional training17.
This work reports a high-speed and open-top UV-PAM system that can be operated similar to conventional optical microscopies, providing both original grayscale histological images and virtually stained images assisted by a deep-learning algorithm. A formalin-fixed and paraffin-embedded (FFPE) mouse brain slice is imaged by the UV-PAM system to demonstrate the similarity between our virtually stained UV-PAM and standard H&amp;E-stained images, showing its potential for SMA applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcohol</td>
			<td>Sigma Aldrich</td>
			<td>PHR1373</td>
			<td>Sample dehydration</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Mini Circuit</td>
			<td>ZFL-500LN-BNC+</td>
			<td>Ultrasonic signal amplification</td>
		</tr>
		<tr>
			<td>Controller</td>
			<td>National Instruments</td>
			<td>NI myRIO</td>
			<td>System controller</td>
		</tr>
		<tr>
			<td>Data acquisition card</td>
			<td>Alazar Technologies</td>
			<td>ATS9350</td>
			<td>Ultrasonic signal collection</td>
		</tr>
		<tr>
			<td>Deep-learning algorithm</td>
			<td></td>
			<td></td>
			<td>For transfering the grayscale UV-PAM image to a virtual H&#38;E-stained image; https://github.com/TABLAB-HKUST/Deep-PAM</td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sigma Aldrich</td>
			<td>R04586</td>
			<td>Sample fixation</td>
		</tr>
		<tr>
			<td>H&#38;E staining kit</td>
			<td>Abcam</td>
			<td>ab245880</td>
			<td>Sample staining</td>
		</tr>
		<tr>
			<td>Histo-Clear II</td>
			<td>National Diagnostics</td>
			<td>HS-202</td>
			<td>Sample deparaffinization</td>
		</tr>
		<tr>
			<td>Image acquisition program</td>
			<td>National Instruments</td>
			<td>LabVIEW</td>
			<td>Lab-built program using LabVIEW; https://github.com/TABLAB-HKUST/LabVIEW-program-for-UV-PAM</td>
		</tr>
		<tr>
			<td>Image processing algorithm</td>
			<td>Mathworks</td>
			<td>MATLAB</td>
			<td>Lab-built algorithm using MATLAB; https://github.com/TABLAB-HKUST/ImageRec_GM-UVPAM</td>
		</tr>
		<tr>
			<td>Kinematic platform mounts</td>
			<td>Thorlabs</td>
			<td>KM200B</td>
			<td>Adjust the sample to be flat</td>
		</tr>
		<tr>
			<td>Membrane</td>
			<td>Glad</td>
			<td>Cling wrap</td>
			<td>Sandwiched in sample tank</td>
		</tr>
		<tr>
			<td>Microscope objective lens</td>
			<td>Thorlabs</td>
			<td>LMU- 5X-NUV</td>
			<td>Objective lens</td>
		</tr>
		<tr>
			<td>Motorized stages</td>
			<td>Physik Instrumente</td>
			<td>L-509.10SD00</td>
			<td>Scanning stages</td>
		</tr>
		<tr>
			<td>One-dimensional galvanometer mirror</td>
			<td>Thorlabs</td>
			<td>GVS411</td>
			<td>Fast scanning mirror</td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>RIGOL Technologies</td>
			<td>DS1102E</td>
			<td>Ultrasonic signal readout</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P3813</td>
			<td>Sample washing</td>
		</tr>
		<tr>
			<td>Pinhole</td>
			<td>Edmund Optics</td>
			<td>#59&#8211;257</td>
			<td>Spatial filtering</td>
		</tr>
		<tr>
			<td>Plano convex lens</td>
			<td>Thorlabs</td>
			<td>LA-4600-UV</td>
			<td>Focusing lens</td>
		</tr>
		<tr>
			<td>Plano convex lens</td>
			<td>Thorlabs</td>
			<td>LA-4663-UV</td>
			<td>Collimating lens</td>
		</tr>
		<tr>
			<td>Pulser/receiver</td>
			<td>Imaginant</td>
			<td>DPR300</td>
			<td>Pulse echo amplifier</td>
		</tr>
		<tr>
			<td>Q-switch diode-pumped solid-state laser</td>
			<td>Bright Solutions</td>
			<td>WEDGE HF 266 nm</td>
			<td>266-nm laser</td>
		</tr>
		<tr>
			<td>Ring-shaped ultrasonic transducer</td>
			<td>University of Southern California</td>
			<td></td>
			<td>Ultrasonic signal detection</td>
		</tr>
		<tr>
			<td>Sample holder</td>
			<td>Lab-made</td>
			<td></td>
			<td>Hold the sample tank</td>
		</tr>
		<tr>
			<td>Sample tank</td>
			<td>Lab-made</td>
			<td></td>
			<td>Hold biological samples</td>
		</tr>
		<tr>
			<td>Single-axis Z-translational stage</td>
			<td>Thorlabs</td>
			<td>PT1</td>
			<td>Manual stage</td>
		</tr>
		<tr>
			<td>Two-axis manual stage</td>
			<td>Thorlabs</td>
			<td>LX20</td>
			<td>Manual stage</td>
		</tr>
		<tr>
			<td>Water tank</td>
			<td>Lab-made</td>
			<td></td>
			<td>Ultrasonic signal transmission</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma Aldrich</td>
			<td>XX0060</td>
			<td>Sample clearing</td>
		</tr>
	</tbody>
</table>

high-speed ultraviolet photoacoustic microscopy for histological imaging with virtual-staining assisted by deep learning

Surgical margin analysis (SMA), an essential procedure to confirm the complete excision of cancerous tissue in tumor resection surgery, requires intraoperative diagnostic tools to avoid repeated surgeries due to a positive surgical margin. Recently, by taking the advantage of the high intrinsic optical absorption of DNA/RNA at 266 nm wavelength, ultraviolet photoacoustic microscopy (UV-PAM) has been developed to provide high-resolution histological images without labeling, showing great promise as an intraoperative tool for SMA. To enable the development of UV-PAM for SMA, here, a high-speed and open-top UV-PAM system is presented, which can be operated similarly to conventional optical microscopies. The UV-PAM system provides a high lateral resolution of 1.2 &#181;m, and a high imaging speed of 55 kHz A-line rate with one-axis galvanometer mirror scanning. Moreover, to ensure UV-PAM images can be easily interpreted by pathologists without additional training, the original grayscale UV-PAM images are virtually stained by a deep-learning algorithm to mimic the standard hematoxylin- and eosin-stained images, enabling training-free histological analysis. Mouse brain slice imaging is performed to demonstrate the high performance of the open-top UV-PAM system, illustrating its great potential for SMA applications.

Figure 1 shows the schematic of the high-speed UV-PAM system. In this setup, the optical excitation and ultrasonic detection paths are on the same side and below the sample, forming a reflective mode and open-top system. Thus, it is user-friendly and suitable for imaging thick samples.
Figure 2 shows the scanning trajectory of the UV-PAM system during imaging. Sub-image of each section (e.g., area of 5 mm x 30 &#181;m) is first generated using a scattered interpolation algorithm, and then, a whole image (e.g., area of 5 mm x 5 mm) is obtained by stitching all the sub-images using custom image processing algorithm (see Table of Materials).
Figure 3A and Figure 3B show the UV-PAM and H&#38;E images of an FFPE mouse brain slice, respectively, both of which have a field-of-view of 5 x 5 mm2. The image processing algorithm can be accessed from Github via the link provided in the Table of Materials. The image acquisition time was less than 18 min. Figure 3C-F show the zoomed-in images of the marked regions in Figure 3A, where the individual cell nuclei can be clearly resolved. More importantly, the corresponding cell nuclei can be found in the standard H&#38;E-stained images (Figure 3G-J), showing the high accuracy of the current system for cellular imaging. To transfer the grayscale UV-PAM image to a virtual H&#38;E-stained image, a deep-learning algorithm17 (see Table of Materials) was applied, which would take less than 30 s for an image with 8000 x 8000 pixels. The corresponding zoomed-in images are shown in Figure 3K-N. The virtually stained UV-PAM images provide almost the same structural information as the H&#38;E-stained images, showing promise for clinical translation of the current UV-PAM system.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63649/63649fig01.jpg" /> 
Figure 1: The schematic of the high-speed and open-top UV-PAM system. (A) The system setup. (B) Photograph of the sample holder and a water tank that is attached to a two-axis manual stage. (C) Photograph of the ring-shaped ultrasonic transducer fixed in the water tank and the sample tank that covers the hole of the sample holder. (D) Photograph of the sample tank with the membrane and sample that would be placed on the sample holder. UV: Ultraviolet; DAQ: Data acquisition card; GM: Galvanometer mirror. <a href="https://www.jove.com/files/ftp_upload/63649/63649fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63649/63649fig02.jpg" /> 
Figure 2: The scanning trajectory of the UV-PAM system during imaging. <a href="https://www.jove.com/files/ftp_upload/63649/63649fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63649/63649fig03.jpg" /> 
Figure 3: Experimental results of UV-PAM imaging with deep learning-based virtual staining. (A) UV-PAM image of an FFPE mouse brain slice. (B) Standard H&#38;E-stained image of the same slice. Scale bars: 1 mm. (C-F) Zoomed-in images of the marked regions in A. (G-J) Corresponding H&#38;E-stained images of the marked regions in B. (K-N) Corresponding virtually stained images using a deep-learning algorithm. Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63649/63649fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High-Speed Ultraviolet Photoacoustic Microscopy for Histological Imaging with Virtual-Staining assisted by Deep Learning at JoVE.com

High-Speed Ultraviolet Photoacoustic Microscopy for Histological Imaging with Virtual-Staining assisted by Deep Learning

A high-speed and open-top ultraviolet photoacoustic microscope that can provide histological images intraoperatively for surgical margin analysis is demonstrated, including the system configuration, optical alignment, sample preparation, and experimental procedures.

Surgical margin analysis (SMA), an essential procedure to confirm the complete excision of cancerous tissue in tumor resection surgery, requires ...

high-speed-ultraviolet-photoacoustic-microscopy-for-histological

Research

JoVE Journal

Bioengineering

2.8K Views.  Hong Kong University of Science and Technology. A high-speed and open-top ultraviolet photoacoustic microscope that can provide histological images intraoperatively for surgical margin analysis is demonstrated, including the system configuration, optical alignment, sample preparation, and experimental procedures.

Video: High-Speed Ultraviolet Photoacoustic Microscopy for Histological Imaging with Virtual-Staining assisted by Deep Learning

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 ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

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., ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a non-contact measurement method that can be used to obtain direct readouts of viscoelastic material properties. It has been a useful tool in material characterization, structural monitoring and environmental sensing. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis. This process requires high illumination power and long acquisition times, making the technique unsuitable for biomedical applications. A recently introduced novel spectrometer overcomes this challenge by employing two VIPAs in a cross-axis configuration. This innovation enables sub-Gigahertz (GHz) resolution spectral analysis with sub-second acquisition time and illumination power within the safety limits of biological tissue. The multiple new applications facilitated by this improvement are currently being explored in biological research and clinical application.

Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

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

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

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

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

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

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, ...