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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

1.7K 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

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

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

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

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

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

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 ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

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

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

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

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

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...