This research is funded by ALIES and COFAC principal providers of the technology under study, and by Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT) through the grant UIDB/04567/2020 to CBIOS.

The experimental protocol was previously approved by the Ethics Committee of the University&#39;s School of Health Sciences (EC.ECTS/P10.21). Procedures fully respected the principles of good clinical practice defined for human research13. A convenient sample of six healthy participants of both sexes (n = 3 per sex) with a mean age of 32.8 &#177; 11.9 years old was chosen from the university community. Selected participants were required to be normotensive, non-smokers, and free of any medication or food supplementation. Blood pressure, cardiac frequency, and the Body Mass Index were also registered. All participants were previously informed of the objectives and duration of the study and provided informed written consent.
NOTE: This study was performed using the MSOTAcuity (see Table of Materials), henceforth referenced as the optoacoustic imaging platform.
1. Preparation for Acquisition
NOTE: In the experimental description that follows, screen commands are in Boldface type.
<ol>
	<li>Loading subject information: Turn on the optoacoustic imaging equipment. While the equipment is warming up, introduce the participant information. The main welcome window of the software opens to the Scan overview. Introduce data (including name, the study denomination, personal data, and any relevant observations) after clicking on Patient ID, and finish the application by pressing Select.</li>
	<li>Preset selection: Make sure the Laser is Ready message appears on the equipment screen. Following the warmup time, the laser status bar on the equipment screen must change from Laser Standby to Laser is Ready. For this protocol, the preset is designed for chromophores Hb, HbO2, and melanin. After selecting the correct preset, the laser power will be tested.</li>
	<li>At this point, ensure that there is a message on the screen that reminds every participant in the room to apply laser safety googles. Press the laser (power) switch foot pedal and wait for the laser power self-check. After a few seconds, a window appears with the current laser status with a check-up report. Release this window by pressing the available OK button. 
	NOTE: The optoacoustic imaging platform uses a Nd:YAG laser, considered a class 4 laser particularly dangerous to the human eye. Thus, this laser must be handled with adequate care. 
	&#8203;CAUTION: No acquisition should take place without ensuring all safety procedures, including appropriate eye protection, are in place.</li>
</ol>
2. Positioning and Image Acquisition
<ol>
	<li>Acclimatize the participant to the laboratory environment (21 &#176;C &#177; 1 &#176;C; 40%-60% relative humidity), choosing a comfortable position to minimize unnecessary movement. Ensure that the area to be scanned is previously cleaned. 
	NOTE: The manufacturer&#39;s recommendation to clean the area to be imaged with a 70% ethanol/water solution is recommended. Additionally, for best image acquisition, removal of hair (when applicable) is suggested.</li>
	<li>Probe holder and image stabilization
	<ol>
		<li>Apply a thin layer of ultrasound gel to the 3D cup. Image stabilization requires holding the 3D cup in the desired imaging position. Position and stabilize a lockable arm for the area of interest. The arm used in this study was designed in-house and built with aluminum profile components (Figure 1).</li>
		<li>After placing the 3D cup on the area of interest, partially lock the stabilizing arm lock for image acquisition. 
		NOTE: The quality and even application of the ultrasound gel is critical; the presence of air-bubbles may compromise the image definition.</li>
	</ol>
	</li>
	<li>Image acquisition for dynamic conditions, using the Post-Occlusive Reactive Hyperemia (PORH) maneuver, in the Examination menu tab.
	<ol>
		<li>Acquire the baseline control scan. After finding a field of view for imaging, with the deflated blood pressure cuff in place, securely lock the 3D cup positioning arm.</li>
		<li>Apply minimal pressure to the imaging site, as higher pressures might compromise the readouts. Push the manufacturer&#39;s default preset Hb, HbO2, and Melanin that simultaneously measures chromophores for Hb, HbO2, and melanin. 
		CAUTION: It is mandatory to protect the eyes with proper safety goggles during operation. 
		NOTE: Skin phototypes IV to VI (dark skin) are prone to misreading, requiring a baseline control image for further processing. The use of safety goggles during image acquisition (when the laser is active) only allows the human eye to recognize yellow and blue colors. Colors can be edited during image processing.</li>
		<li>Select the anatomical area for baseline image acquisition. For exploratory purposes, the ventral forearm is recommended. Proceed by pressing the laser footswitch pedal. 
		NOTE: The touch screen button labeled View (colored yellow), which shows the live image on the screen when pressed. The image stability status is shown as a grey bar in the middle of the touch screen, indicating the stability of the 3D probe.
		<ol>
			<li>When the image stability is maximized, take (or capture) a snapshot of the area by pressing the Snapshot button on the touchscreen. Each scan will acquire 10-12 frames at an acoustic depth of 150 mm for every wavelength defined within the preset over an acquisition time of 2 s. This baseline acquisition scan will include a total of 30-36 frames. 
			NOTE: 10 frames for each chromophore detected (Hb, HbO2, and melanin) are collected with a maximal depth of 15 mm.</li>
			<li>Continue pressing the laser footswitch pedal for continuous video acquisition and pay attention to the View button (colored yellow) on the touch screen. The stabilized image will appear. Press Record (colored blue) to begin live image recording.</li>
			<li>Stop the recording by pressing the Stop button (colored black). The optoacoustic imaging platform will stop recording and automatically render the video to preview mode.</li>
		</ol>
		</li>
		<li>Dynamic measurements (PORH illustration): Adjust the pressure cuff to the patient&#39;s arm above the elbow to illustrate this maneuver. Inflate the cuff with supra systolic pressure (~200 mmHg) and proceed according to steps 2.3.1 to 2.3.3.1 to acquire the imaged vasculature under pressure.</li>
		<li>To acquire a video to assess the impact of the pressure release on the imaged vasculature, open the pressure valve while acquiring the video as in 2.3.3.2. As before, follow the live image on the screen. 
		NOTE: To execute this maneuver, supra systolic pressure should be maintained for 1-5 min; it is important to be aware that this pressure might induce different degrees of tolerance and discomfort in the patient. This aspect should be carefully managed during the experiments.</li>
	</ol>
	</li>
</ol>
3. Image analysis protocol
<ol>
	<li>Copy the recorded scans to a selected/dedicated folder for backup and further analysis on a separate computer workstation using the manufacturer&#39;s dedicated analytical software. Each scan is stored by acquisition time and ordered by the program in a study folder with a running code. 
	NOTE: A backup copy is strongly recommended. Working directly with the recorded raw data is possible but strongly discouraged, as any potential hard drive crash might damage the raw data.</li>
	<li>Open the analysis program on the workstation computer. Choose program Menu &#62; Open Study to import files and access backup scans. Open the study and scroll to the bottom of the&#160;folder (with recorded scans) to find files with a .NOD extension. This is the only file type recognized by the software to open a study. 
	NOTE: .NOD files are named automatically with a running number given to each study and carry no patient information in the file name.</li>
	<li>For image reconstruction, open the image analysis module by accessing the software Menu &#62; Advanced processing.
	<ol>
		<li>Ensure that the program workflow tabs are visible (colored black) on the top menu bar (Supplementary Figure 1): Menu; Scan Overview; Reconstruction; Fluence Correction; Spectral Unmixing; Visualization &#38; Analysis. During analysis, any activated workflow tab is colored blue. 
		NOTE: If Advance Processing is not opened, the software shows only Scan Overview and Visualization &#38; Analysis.</li>
	</ol>
	</li>
	<li>Reconstruct the image via the Reconstruction tab of the software. Select the scans to reconstruct from the left side of the main program menu. Loaded&#160;scans appear on the right side of the screen. Leave the default six optoacoustic emission wavelengths (700, 730, 760, 800, 850, and 900 nm), as they include the maximum optoacoustic signal for HbO2 at 900 nm, for Hb at 760 nm, and melanin at 700 nm.
	<ol>
		<li>Perform the scan reconstruction using the icon on the right side. Follow the program workflow by selecting the Scan preset and Field of View (resolution). Information is presented at the top left corner of the main screen. Adjust the speed of sound to adjust scan focus (Supplementary Figure 2). The Reconstruction panel also shows the number of frames of each acquired scan and allows the selection of the repetitions to be analyzed (if necessary). 
		NOTE: Each scan is loaded with a default speed of sound of -90, which should be adjusted by the user. The speed of sound may also be adjusted automatically with an auto-focus function (AF).</li>
	</ol>
	</li>
	<li>Push the button Reconstruct Scans at the top of the screen to advance to scan reconstruction. A temporary dashboard will appear with the message Job Processing. This panel can also be accessed from the Menu &#62; Processing Status. After finishing the reconstruction, the image post-processing analysis must advance to Fluence Correction.</li>
	<li>Activate Fluence Correction of reconstructed images in the dashboard menu. Reconstructed images must be loaded for fluence correction. These appear with a flag next to each scan number. Loaded files will be immediately displayed on the right side of the screen as Selected Reconstructions. Activate Fluence Correction by interacting with the icon on the right side of the screen (Supplementary Figure 3). Push the Save Fluence Correction(s) to progress.</li>
	<li>After saving the fluence correction, perform the spectral unmixing of the acquired preset (Hb, HbO2, and melanin). Select the Spectral Unmixing tab to open the list of Selected Reconstructions for spectral unmixing. A list with each scan of the selected study will be displayed with the history of the previous image processing steps.</li>
	<li>Load the previously saved fluence correction files. Loaded scans will be immediately displayed on the right side of the screen as Selected Reconstructions (Supplementary Figure 4). Activate spectral unmixing by pushing the icon on the right side of the screen.
	<ol>
		<li>Observe the wavelengths to be unmixed. All six optoacoustic emission wavelengths (700, 730, 760, 800, 850, and 900 nm) taken into the reconstruction step (step 3.4) are automatically chosen for spectral unmixing. Edit the desired spectra to be processed (e.g., Spectra: Hb, HbO2, and melanin) using the XYZ icon, if necessary.</li>
		<li>After confirming the adjusted parameters, click on Start Spectral Unmixing for the spectral unmixing to progress. A processing menu bar appears, displaying the operation progress. 
		NOTE: Various parameter adjustments are possible during spectral unmixing, and several unmixing methods are available. In this protocol, the Linear Regression method is used as a standard to unmix Hb, HbO2, and melanin.</li>
	</ol>
	</li>
	<li>Access the Visualization and Analysis tab. Click an activated scan to display all subject information and comments introduced in step 1.1 (Supplementary Figure 5). 
	NOTE: Multiple scans may be visualized in parallel.
	<ol>
		<li>Push the + button to create a multiple scan analysis. In this window, introduce a multiple scan view and press the Save button. After saving the view name, a new dashboard is displayed, including all the scans of the study being analyzed.</li>
		<li>Select each desired scan to Add (each) to the saved Analysis View. Add additional scans at the top left corner icon, and they will be automatically displayed in the Analysis View.</li>
	</ol>
	</li>
	<li>Within the analysis view, set proper color lookup tables to prepare the image for analysis. Click More Image Control Options on the top menu bar and activate the Max Intensity Projection icon. Attribute colors to layers by pressing the icon available in the bottom right corner of the image display, adjacent to the 2D+ image display.</li>
	<li>Select More to edit the colors of all channels simultaneously. This menu shows all chromophores unmixed and allows the selection of multiple layers for display. 
	NOTE: Moving the mouse over the software icons shows their name in gray as displayed in the protocol.</li>
	<li>Adjust each layer&#39;s color intensity with the tools available at the bottom left of the screen. 
	&#8203;NOTE: Adjustment with min/max interpolation for each channel generally yields good results.</li>
</ol>
4. Region of Interest (ROI) analysis
NOTE: The selection of a Region of Interest (ROI) is mandatory for data analysis.
<ol>
	<li>Identify the ROI to be analyzed. Surround the ROI with the shapes available (within the menu bar) in the XY image while tracing the same ROI within the orthogonal views available in the XZ and YZ axis (Supplementary Figure 6). 
	NOTE: A polygon shape was used for the current ROI analysis.
	<ol>
		<li>Follow the ROI shape in the remaining XZ and YZ axis (example in Figure 2) while placing multiple polygon layers with the Add Interpolate and Remove sub-regions function. The data can be plotted after defining/selecting the desired ROI.</li>
		<li>Press the icon Import Region of Interest to Quantification and observe the multi spectra component shown on the right side of the screen as a graphical detail of the selected ROI.</li>
		<li>Export ROI data by pressing the Excel icon at the bottom of the graphical view of the ROI data. The entire data package from all regions is exported as a bundle to a spreadsheet for subsequent analysis. Figure 3 shows data from one participant who submitted to a pressure cuff inflated to 200 mmHg and vasculature was analyzed in comparison to vasculature resting state at 0 mmHg.</li>
	</ol>
	</li>
	<li>Quantify multiple ROI objects simultaneously by following steps 4.1-4.1.3.</li>
	<li>Export images from the same menu as TIFF files with all embedded data and built-in ROI outline (Figure 2).</li>
</ol>

<ol>
	<li>Iskander-Rizk, S., vander Steen, A. F. W., van Soest, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+imaging+for+guidance+of+interventions+in+cardiovascular+medicine.">Photoacoustic imaging for guidance of interventions in cardiovascular medicine.</a> Physics in Medicine and Biology. 64 (16), (2019).</li><li>Cakmak, H. A., Demir, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+and+cardiovascular+diseases.">MicroRNA and cardiovascular diseases.</a> Balkan Medical Journal. 37 (2), 60-71 (2020).</li><li>Li, Z., Gupte, A. A., Zhang, A., Hamilton, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pet+imaging+and+its+application+in+cardiovascular+diseases.">Pet imaging and its application in cardiovascular diseases.</a> Methodist DeBakey Cardiovascular Journal. 13 (1), 29-33 (2017).</li><li>Karlas, A., 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=Cardiovascular+optoacoustics:+From+mice+to+men+-+A+review.">Cardiovascular optoacoustics: From mice to men - A review.</a> Photoacoustics. 14, 19-30 (2019).</li><li>MacRitchie, N., Noonan, J., Guzik, T. J., Maffia, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+cardiovascular+inflammation.">Molecular imaging of cardiovascular inflammation.</a> British Journal of Pharmacology. 178 (21), 4216-4245 (2021).</li><li>Granja, T., Andrade, S., Rodrigues, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optoaccoustic+tomography+-+good+news+for+microcirculatory+research.">Optoaccoustic tomography - good news for microcirculatory research.</a> Biomedical and Biopharmaceutical Research. 18 (2), 1-13 (2022).</li><li>Tan, H., 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=Total-body+PET/CT:+Current+applications+and+future+perspectives.">Total-body PET/CT: Current applications and future perspectives.</a> American Journal of Roentgenology. 215 (2), 325-337 (2020).</li><li>Masthoff, M., 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=Multispectral+optoacoustic+tomography+of+systemic+sclerosis.">Multispectral optoacoustic tomography of systemic sclerosis.</a> Journal of Biophotonics. 11 (11), 201800155 (2018).</li><li>Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+imaging+and+characterization+of+the+microvasculature.">Photoacoustic imaging and characterization of the microvasculature.</a> Journal of Biomedical Optics. 15 (1), 011101 (2010).</li><li>Wu, M., Awasthi, N., Rad, N. M., Pluim, J. P. W., Lopata, R. G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+ultrasound+and+photoacoustic+imaging+in+cardiology.">Advanced ultrasound and photoacoustic imaging in cardiology.</a> Sensors (Basel). 21 (23), 7947 (2021).</li><li>Yang, H., 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=Soft+ultrasound+priors+in+optoacoustic+reconstruction:+Improving+clinical+vascular+imaging.">Soft ultrasound priors in optoacoustic reconstruction: Improving clinical vascular imaging.</a> Photoacoustics. 19, 100172 (2020).</li><li>Dean-Ben, X. L., Gottschalk, S., Mc Larney, B., Shoham, S., 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=Advanced+optoacoustic+methods+for+multiscale+imaging+of+in+vivo+dynamics.">Advanced optoacoustic methods for multiscale imaging of in vivo dynamics.</a> Chemical Society Reviews. 46 (8), 2158-2198 (2017).</li><li>World Medical Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=World+Medical+Association+Declaration+of+Helsinki:+ethical+principles+for+medical+research+involving+human+subjects.">World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects.</a> JAMA. 310 (20), 2191-2194 (2013).</li></ol>

The authors report no conflicts of interest.

This protocol emphasizes the working steps regarded as practical requirements to operate this new optoacoustic imaging instrument, from the adequate positioning (participant, probe) needed for 3D cup probe stabilization to image acquisition, ROI selection, and image reconstruction and analysis.
The proposed experimental approach, using &#34;instantaneous&#34; acquisitions together with images obtained under dynamic conditions, illustrates the interest and utility of this instrument in accessin...

Cardiovascular diseases are recurrent top causes of death worldwide and represent a huge burden for any health system1,2. Technology has been a major contributor to the expansion of our understanding of fundamental cardiac and vascular pathophysiology, providing more precise diagnostic tools and the possibility of early disease detection and more effective management. Imaging techniques offer the possibility to measure not only cardiac and major vessel performance but also, on a much smaller scale, to calculate the capillary density, local perfusion and volume, and endothelial dysfunction, among other characteristics. These technologies have offered the first quantitative insights into vascular biology with direct clinical application. Changes in capillary density, local perfusion reduction, or occlusion likely correspond to an ischemic condition, which helps to explain the growing role of imaging, becoming an indispensable tool in cardiovascular research and practice3,4,5.
In recent years, functional imaging has consistently set the pace in technological innovation, with ultrasound (US) near-infrared spectroscopy (NIRS), positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) as some well-known examples. However, multiple concerns limit their application, from cost and patient safety (as well as comfort) to image contrast and resolution6,7. Optoacoustic tomography (OT) has recently emerged as a new direction in optical-based vascular research. This technology, centered on the detection of ultrasonic waves generated by thermoelastic expansion of the tissue impacted with ultrashort laser pulses, has been known for some time6,8. This physical reaction of heat development and tissue expansion evokes an acoustic signal detected by an ultrasound transducer. The use of pulses of light from visible to near-infrared and the absence of an acoustic background signal benefit the resolution depth. The detected contrast results from the most important chromophores present (hemoglobin or melanin). Compared with other technologies, OT has the advantages of (1) needing no contrast (label-free imaging), (2) better contrast and resolution with fewer artifacts than ultrasonography, and (3) lower price, and faster acquisition and ease of operation6,9,10,11.
Multispectral optoacoustic tomography (MSOT) is among the most recent generation of OT instruments. Built with a tunable optical parametric oscillator (OPO) pumped by an Nd:YAG laser providing excitation pulses, a 3D image is acquired by time-resolved signals detected from high-frequency ultrasonic excitation pulses at wavelengths from 680 nm to 980 nm with a repetition rate of up to 50 Hz12. The optoacoustic imaging platform provides the quantification of different chromophores in-depth (as low as 15 mm). Variables such as HbO2, Hb, and melanin are easily accessible. Other variables of interest, such as maximal Oxygenated Hemoglobin (Max HbO2) and Deoxygenated Hemoglobin (Max Hb), are also available. Reconstruction algorithms from the manufacturer&#39;s software allow the calculation of other variables such as vascular density (&#181;Vu), inter-unit average distance (&#950;Ad), and capillary volume (mm3).
The present study explores the essential operating aspects of this new system to understand better its practicalities and potential applications in cardiovascular preclinical research.&#160;

<table><tbody><tr><td>Cuff</td><td>PIC</td><td>107001</td><td></td></tr><tr><td>Drapes</td><td>Pajunk</td><td>021151-1501</td><td></td></tr><tr><td>Ethanol 70%</td><td>Sigma Aldrich</td><td>EX0281</td><td></td></tr><tr><td>Gogless</td><td>Univet</td><td>559G.00.00.201</td><td></td></tr><tr><td>Kimwipes</td><td>Amoos</td><td>5601856202331.00</td><td></td></tr><tr><td>MSOT</td><td>iThera</td><td>MSOTAcuity</td><td></td></tr><tr><td>Stabilizing arm</td><td>ITEM</td><td></td><td>Self designed and assemble</td></tr><tr><td>Ultrasound gel</td><td>Parker Laboratories</td><td>308</td><td></td></tr><tr><td>Waxing cream</td><td>Veet</td><td>kkdg08hagd</td><td></td></tr></tbody></table>

multispectral optoacoustic tomography for functional imaging in vascular research

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the development of live imaging systems has set the (analytical) pace in both basic and clinical research, with the objective of creating new instruments capable of providing real-time, quantifiable endpoints with clinical interest and application. Near-infrared spectroscopy (NIRS), positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) are available, among other techniques, but cost, image resolution, and reduced contrast are recognized as common challenges. Optoacoustic tomography (OT) offers a new perspective on vascular functional imaging, combining state-of-the-art optical absorption and spatial resolution capacities (from micrometer optical to millimeter acoustic resolution) with tissue depth. In this study, we tested the applicability of multispectral optoacoustic tomography (MSOT) for functional imaging. The system uses a tunable optical parametric oscillator (OPO) pumped by an Nd: YAG laser, providing excitation pulses sensed by a 3D probe at wavelengths from 680 nm to 980 nm. Images obtained from the human forearm were reconstructed through a specific algorithm (supplied within the manufacturer&#39;s software) based on the response of specific chromophores. Maximal Oxygenated Hemoglobin (Max HbO2) and Deoxygenated Hemoglobin (Max Hb), Total Hemoglobin (HbT), and mean Oxygen Saturation (mSO2) to vascular density (&#181;Vu), inter-unit average distances (&#950;Ad), and capillary blood volume (mm3) may be measured using this system. The applicability potential found with this OT system is relevant. Ongoing software developments will surely improve the utility of this imaging system.

Data provided by optoacoustic imaging can be analyzed in post-processed export images (Figure 2) and plotted data (Figure 3). The purpose here was to introduce the operation of optoacoustic functional imaging and to explore its application in more commonly known vascular research. For that, we compared images acquired during rest and after a 200 mmHg occlusion of a major supplying artery (Figure 2). These observations can be quantif...

Watch this Scientific Journal Video about Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research at JoVE.com

Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research

The present protocol describes the acquisition of multispectral optoacoustic images of in vivo human skin vasculature. These include the quantification of hemoglobin and melanin, regarded as chromophores of interest for functional analysis.

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the ...

multispectral-optoacoustic-tomography-for-functional-imaging-vascular

Research

JoVE Journal

Bioengineering

1.8K Views.  CBIOS - Universidade Lusófona‘s Research Center for Biosciences and Health Technologies. The present protocol describes the acquisition of multispectral optoacoustic images of in vivo human skin vasculature. These include the quantification of hemoglobin and melanin, regarded as chromophores of interest for functional analysis. 

Video: Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Medicine

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

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

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

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

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

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

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Neuroscience

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 ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

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 ...

Non-Invasive Integrated PAUSAT Whole-Brain Imaging of Ischemic Stroke

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.

Author Spotlight: Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke  

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging ...

Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research

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Chapters in this video

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Three-dimensional Optical-resolution Photoacoustic Microscopy

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

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

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

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

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

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

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke