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 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'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 (µVu), inter-unit average distances (ζAd), 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.
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's software allow the calculation of other variables such as vascular density (µVu), inter-unit average distance (ζAd), 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.
The experimental protocol was previously approved by the Ethics Committee of the University'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 ± 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.
2. Positioning and Image Acquisition
3. Image analysis protocol
4. Region of Interest (ROI) analysis
NOTE: The selection of a Region of Interest (ROI) is mandatory for data analysis.
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 quantified after ROI analysis and export. In the XY plane, the higher signal of melanin compared to planes YZ and XZ can be observed, which indicates the epidermis limit. The occlusion of the brachial artery (arm) provokes some stasis in the vessels ahead of the OT probe placement (ventral forearm). In consequence, we detected an increase in the overall signals shown as an increase of blue (Hb) and red (HbO2) at axes XY, YZ, and XZ. Stasis might be followed in the XY plane while holding the 200 mmHg pressure within the cuff. YZ and XZ axes depict increased blood volume due to the occlusion above compared to the normal perfusion conditions (no occlusion), highlighted by the magenta masked areas.
Exported ROI analysis of the same microvasculature area quantifies chromophores HbO2 (red), Hb (blue) and HbT (pink), mSO2 (deep red), and melanin (yellow) from stabilized images collected over 8.6 s. The pressure release is immediately detected; Figure 3 shows the post-occlusion evolution of Hb, HbO2, and HbT recovery, while the optoacoustic data output follows the observations in Figure 1. The software calculates blood oxygen saturation (mSO2) and HbT values from the addition of the Hb and HbO2 arbitrary signals. Melanin concentration remains constant within the 200 mmHg occlusion and at the resting state within the time interval of image acquisition.
Figure 1: Schematic diagram representing the flexible arm designed to hold the measuring probe in stabilized contact with the participant's skin. Please click here to view a larger version of this figure.
Figure 2: Representative optoacoustic images highlighting changes of the vasculature at rest or under pressure of 200 mmHg. The image shown includes three colors, representing Hb (blue), HbO2 (red), and melanin (yellow), as described within the image analysis in section 4. Each optoacoustic image represents a maximum intensity projection of all the planes associated with each scanned chromophore. (A) The XY plane of the optoacoustic acquisition. (B) The YZ orthogonal view of the same optoacoustic imaged site. (C) The XZ view of the scanned area. The magenta arrows point to the areas with increased stasis; the magenta masked area marks the increased volume of blood trapped inside vessels due to the occlusion of the brachial artery compared to the normal perfusion conditions (no occlusion). Please click here to view a larger version of this figure.
Figure 3: Representative data export of a quantified ROI. Natural chromophores of HbO2 (Red), Hb (Blue) and HbT (Pink), mSO2 (deep red), and melanin (yellow) are depicted from the data extracted from the stabilized images collected over 8.6 s. The graphs from Hb, HbO2, and HbT show a recovery slope from occlusion towards the non-occluded resting state. The calculated blood oxygenation mSO2 and the melanin concentration remain constant within the 200 mmHg occlusion and at the resting state within the time interval of image acquisition. Images extracted are data points depicted as mean ± sd of n = 10 images per frame. Please click here to view a larger version of this figure.
Supplementary Figure 1: Scan overview panel and Main Menu of the analysis software. Pushing the Menu button (in black), the main menu will drop down options to select the selected study. This action will select and load the ".nod" file recognised by the software. The Scan overview (in blue) shows all study's scans. Details (black) appear in the right. Please click here to download this File.
Supplementary Figure 2: Reconstruction analysis workflow. Panel 1 – Select the scan to be reconstructed and press the right-facing arrow on the right side of the display (purple arrow) to advance. Panel 2 – Observe the speed of sound and adjust the slider to the best focus (blue arrow); a) adjusted focus displayed in the window right hand side; b) select repetitions to be analysed (yellow arrow); c) press the Reconstruct scans button to proceed (green arrow). Please click here to download this File.
Supplementary Figure 3: Fluence correction panel Workflow. Panel 1 – Select scans to be corrected and press the right-facing arrow on the right side of the screen. Panel 2 – press Save Fluence Correction(s) to proceed (green arrow). Please click here to download this File.
Supplementary Figure 4: Spectra unmixing panel Workflow. Panel 1 – Select scans to unmix and press the right-facing arrow (purple arrow). Panel 2 a) select the scan to unmix (blue arrow) and a preview of the adjusted image will be displayed in the right hand side; b) Select the repetitions to unmix (yellow arrow); c) press Start spectral unmixing to proceed (green arrow). Please click here to download this File.
Supplementary Figure 5: Visualization panel and selection of chromophore colors. Panel 1) Select scans to display with a double click (purple arrow); Panel 2) Acquired image in axis XY (blue square), XZ (yellow square), and YZ (green square); 2a) Image analysis button showing acquired wavelengths; 2b) choose More Image Control Options in the top menu bar and activate the Max Intensity Projection icon; select Plus to edit channels' colors. Please click here to download this File.
Supplementary Figure 6: Selection of the Region of Interest (ROI). Select the lasso tool (yellow arrow) and define the boundaries of the ROI within the XY axis (magenta arrow). It is possible to define various shape areas (polygon, rectangle, square, circle, or elipse). Follow the ROI in XZ and YZ axis and add sub-regions (green arrow) to the initial selection. Multiple subregions are displayed (cyan arrow). To extract data from the selected ROI, press the icon Import Region of Interest to Quantification and proceed. Please click here to download this File.
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 "instantaneous" acquisitions together with images obtained under dynamic conditions, illustrates the interest and utility of this instrument in accessing in vivo human vascular physiology. As shown, the 150 µm acoustic image resolution collected in a volume of up to 15 mm3 is unmatched by other tomography techniques.
Special attention is necessary regarding (i) the importance of the probe stabilization for image acquisition; the use of a flexible, secure probe holder clearly improves image acquisition; (ii) the correct identification of the vascular structures; sonographic references such as melanin in the epidermal-dermal transition might be used as a marker to identify the upper plexus vessels in the skin; and (iii) the functional image analysis performed through the manufacturer´s reconstruction software.
Advanced analysis of ROI data and image export requires a deeper comprehension of the dedicated software and the algorithms developed. The current optoacoustic imaging instrument is able to reconstruct a 3D volume of 15 mm3 of tissue with a resolution of 150 µm. This capacity should be potentiated to better quantify microvascular function(s) in depth. Nevertheless, the basic operation allows the direct observation of reference chromophores and the acquisition of multiple presets from the same area, providing fast scanning and live video recordings.
The applicability potential found with the optoacoustic imaging system is relevant. Ongoing software developments will surely improve the utility of this imaging system.
The authors have nothing to disclose.
This research is funded by ALIES and COFAC principal providers of the technology under study, and by Fundação para a Ciência e a Tecnologia (FCT) through the grant UIDB/04567/2020 to CBIOS.
Cuff | PIC | 107001 | |
Drapes | Pajunk | 021151-1501 | |
Ethanol 70% | Sigma Aldrich | EX0281 | |
Gogless | Univet | 559G.00.00.201 | |
Kimwipes | Amoos | 5601856202331.00 | |
MSOT | iThera | MSOTAcuity | |
Stabilizing arm | ITEM | Self designed and assemble | |
Ultrasound gel | Parker Laboratories | 308 | |
Waxing cream | Veet | kkdg08hagd |