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><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&amp;amp;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&amp;amp;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&amp;ndash;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

2.9K Views.  Hong Kong University of Science and Technology. Intraoperatieve chirurgische monitoranalyse is nog steeds een grote uitdaging bij tumorresectieoperaties omdat het een diagnostisch hulpmiddel vereist om de complexe excisie van kankerweefsel nauwkeurig en snel te bevestigen. Het is dan mogelijk om herhaalde operaties te voorkomen vanwege een positieve chirurgische marge. Onze snelle open-top ultraviolette fotoakoestische microscopie kan binnen 18 minuten gelabelde histologische beelden van weefselmonsters leveren met een beeldvormingsgebied ...

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

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Cancer Research

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 ...

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

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

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

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

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Immunology and Infection

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

Engineering

Label-free Super-resolution Imaging Enabled by Vibrational Imaging of Swelled Tissue and Analysis

The universal utilization of fluorescence microscopy, especially super-resolution microscopy, has greatly advanced knowledge about modern biology. Conversely, the requirement of fluorophore labeling in fluorescent techniques poses significant challenges, such as photobleaching and non-uniform labeling of fluorescent probes and prolonged sample processing. In this protocol, the detailed working procedures of vibrational imaging of swelled tissue and analysis (VISTA) are presented. VISTA circumvents obstacles associated with fluorophores and achieves label-free super-resolution volumetric imaging in biological samples with spatial resolution down to 78 nm. The procedure is established by embedding cells and tissues in hydrogel, isotropically expanding the hydrogel sample hybrid, and visualizing endogenous protein distributions by vibrational imaging with stimulated Raman scattering microscopy. The method is demonstrated on both cells and mouse brain tissues. Highly correlative VISTA and immunofluorescence images were observed, validating the protein origin of imaging specificities. Exploiting such correlation, a machine learning-based image-segmentation algorithm was trained to achieve multi-component prediction of nuclei, blood vessels, neuronal cells, and dendrites from label-free mouse brain images. The procedure was further adapted to investigate pathological poly-glutamine (polyQ) aggregates in cells and amyloid-beta (A&#946;) plaques in brain tissues with high throughput, justifying its potential for large-scale clinical samples.

By combining sample-expansion hydrogel chemistry with label-free chemical-specific stimulated Raman scattering microscopy, the protocol describes how to achieve label-free super-resolution volumetric imaging in biological samples. With an additional machine learning image segmentation algorithm, protein-specific multi-component images in tissues without antibody labeling were obtained.

The universal utilization of fluorescence microscopy, especially super-resolution microscopy, has greatly advanced knowledge about modern biology. ...

Chemistry

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, ...

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

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

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

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