Name: integrated photoacoustic, ultrasound, and angiographic tomography (pausat) for noninvasive whole-brain imaging of ischemic stroke
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The authors would like to acknowledge the engineering team at SonoVol Inc. for their technical support. This work was partially sponsored by the American Heart Association Collaborative Sciences Award (18CSA34080277), to J. Yao and W. Yang; The United States National Institutes of Health (NIH) grants R21EB027981, R21 EB027304, RF1 NS115581 (BRAIN Initiative), R01 NS111039, R01 EB028143; The United States National Science Foundation (NSF) CAREER award 2144788; the Chan Zuckerberg Initiative Grant (2020-226178), to J. Yao; and NIH grants R21NS127163 and R01NS099590 to W. Yang.

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

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The authors declare no conflict of interest in this work.

There are a few vital aspects of this method that, if done incorrectly, can lead to significantly decreased image quality and quantitative analysis. The most commonly occurring result of user-error in PAUSAT images is either a lack of signal or very low signal strength, both of which can occur for a variety of reasons. One such reason is a problem with the acoustic coupling. Large air bubbles in the water surrounding the mouse&#39;s head during imaging can often block the ultrasound from travelling to or from the transducer, causing a shadow region in the resulting image for all three modalities of the system. This can be prevented by ensuring that enough water is present between the system membrane and the sample to be imaged. It could also occur that there is a lack of AA signal, but a surprisingly high PA signal, independent of the wavelengths used. This could be due to the presence of hair-especially dark hair-interfering with the absorption of the light. To avoid this, it is necessary to have previously shaved the animal&#39;s head until no hair is visually identifiable.
Regarding AA images, another problem that might occur and would lead to the lack of or very low signal is a low concentration of microbubbles present in the circulatory system. If the microbubbles are too diluted or they were injected improperly, the resulting signal will be very weak. The retro-orbital injection must be performed only by well-trained personnel. Another reason could be a long period of time between microbubble injection and the start of imaging, which can lead to a reduction of the microbubbles in the bloodstream. To avoid this, it is recommended to prepare the system for imaging before injecting the microbubbles, so the animal can be transferred to the PAUSAT system membrane immediately after injection. It is worth mentioning that alternative intravenous routes (such as tail vein injection) can also be used once the animal is already positioned on the PAUSAT system membrane, shortening the time between injection and imaging. Additionally, in the interest of maintaining the maximum concentration of microbubbles possible, it is also recommended to perform the AA imaging before the PA imaging.
The PA signal can also be affected if the necessary steps are not properly performed. One of the issues involves the quality of the excitation laser. The two main components that describe the laser source are the pulse energy and the wavelength. It is highly recommended to use an independent power meter and spectrometer to measure these quantities and ensure they are set to the desired values. If an incorrect value is assumed, the functional calculations will provide misleading results.
It is also possible that when AA and PA images are combined, they are out of alignment. The main reason for this is that the coordinates established between the transducers were not accurate. To prevent this, it is critical to perform the required experiments using a phantom grid beforehand to determine the exact coordinates for a successful co-registration of AA and PA images. Another potential source of inaccuracy is due to incorrect calibration of the OPO. To mitigate this, the OPO must be correctly calibrated using an independent spectrometer.
There remain significant areas of improvement, particularly regarding the image quality of the PAT component of this integrated system. The current PAT system is based on a scanning linear-array configuration. We are able to observe large-scale differences in the oxygenation of infarct versus healthy regions of the brain using several stroke mouse models, however, the detailed vasculature (microvessels) is not able to be seen. There are two main problems that contribute to this low image quality. The first is the limited-view problem. In order to create a full-view reconstructed image of a sample in PAT, the detector must completely surround the object (or have a solid angle of 4&#960;). However, in the experimental setting, this is difficult. This gives rise to the limited view problem, leading to blood vessels that are orthogonal to the transducer array, being undetectable21. Solutions to reduce the effects of the limited-view problem in linear-array PAT do exist, the most promising of which is the use of microbubbles as virtual point sources22. The second problem of linear-array PAT is poor elevational resolution and sensitivity, due to the weak focus of the acoustic lens. However, the use of single-slit diffraction in the hardware design of a linear-array system has been shown to create isotropic resolution and sensitivity for linear-array PAT23,24. Deep learning approaches have also been shown to partially resolve both the limited-view problem and the poor elevational resolution of linear-array PAT25,26,27. The combination of these solutions would significantly enhance the image quality of the PAT component of our integrated imaging system.
Here, we have presented a novel noninvasive multi-modal imaging method for the structural and functional quantification of ischemic stroke in the preclinical setting. Through AA, clear morphology and perfusion mapping of blood vessels in the mouse brain is possible. A locally defined lack of signal can indicate an infarct region where there is decreased blood perfusion, allowing for the estimation of infarct volume noninvasively and longitudinally. Through PAT, the oxygen saturation of hemoglobin can be measured in- and outside of the stroke region, showing the hypoxic state of brain tissues in the infarct region. This method of whole-brain imaging is low-cost with respect to some alternatives, such as small-animal MRI. Furthermore, it allows for the combination of functional and structural information in deep tissue that other preclinical imaging devices are unable to achieve. Compared to histological analyses, this method allows for measurement of these metrics longitudinally and at multiple time points, as opposed to at a single endpoint. The ability to do so will provide unprecedented quantitative detail in the preclinical study of ischemic stroke. Additionally, the structural and functional imaging capability of this system can be applied to many preclinical studies beyond ischemic stroke28. Any disease state or biological phenomenon affecting the vascular system could be studied in depth using PAUSAT, making it a powerful preclinical imaging tool translatable to many other preclinical fields (e.g., cancer).

Blood transports oxygen (via the hemoglobin protein) and other important nutrients to tissues in our bodies. When the flow of blood through tissues is interrupted (ischemia), severe damage to the tissues can occur, the most immediate effects of which are due to a lack of oxygen (hypoxia). Ischemic stroke is the result of interrupted blood flow to a certain region of the brain. The brain damage resulting from an ischemic stroke can occur within minutes of a vessel blockage, and can often have debilitating and lasting effects1,2. A highly valuable strategy to evaluate the physiopathology after ischemic stroke and identify and test new treatments is the use of small-animal models in the lab. Treatments discovered in the lab aim to be translated to clinical use and improve patients&#39; lives. However, the use of animals in biomedical research needs to be carefully evaluated according to Russell and Burch&#8217;s 3Rs principles: replacement, reduction, and refinement3. The objective of the reduction component is to reduce the number of animals without compromising data collection. With this in mind, being able to longitudinally evaluate the lesion evolution via noninvasive imaging allows a great advantage in reducing the number of animals required, as well as maximizing the information obtained from each animal4.
Photoacoustic tomography (PAT) is a hybrid imaging modality that combines optical absorption contrast with ultrasound imaging spatial resolution5. The imaging mechanism of PAT is as follows. An excitation laser pulse is illuminated on the target being imaged. Assuming the target absorbs light at the wavelength of the excitation laser, it will increase in temperature. This quick increase in temperature results in a thermoelastic expansion of the target. The expansion causes an ultrasound wave to propagate out from the target. By detecting the ultrasound wave at many positions, the time required for the wave to propagate from the target to the detectors can be used to create an image through a reconstruction algorithm. The ability of PAT to detect optical absorption in deep tissue regions differentiates PAT from ultrasound imaging, which detects boundaries of differing acoustic impedances of tissues5. In the visible and near-infrared spectra, the primary highly absorbing biomolecules that are abundant in organisms are hemoglobin, lipids, melanin, and water7. Of particular interest in the study of stroke is hemoglobin. Since oxyhemoglobin and deoxyhemoglobin have different optical absorption spectra, PAT can be used with multiple excitation laser wavelengths to determine the relative concentration of the two states of the protein. This allows the oxygen saturation of hemoglobin (sO2), or blood oxygenation, to be quantified in and outside of the infarct region8,9. This is an important measure in ischemic stroke, as it can indicate the level of oxygen in the damaged brain tissue following ischemia.
Acoustic angiography (AA) is a contrast-enhanced ultrasound imaging method that is particularly useful for imaging the morphology of vasculature in vivo10. The method relies on the use of a dual-element wobbler transducer (a low frequency element and a high frequency element) in conjunction with microbubbles injected into the circulatory system of the imaging subject. The low-frequency element of the transducer is used for transmitting at the resonant frequency of the microbubbles (e.g., 2 MHz), while the high-frequency element is used to receive the super harmonic signals of the microbubbles (e.g., 26&#160;MHz). When excited at a resonant frequency, the microbubbles have a strong nonlinear response, resulting in the production of super harmonic signals that surrounding body tissues do not produce11. By receiving with a high-frequency element, this ensures that only the microbubble signals are detected. Since the microbubbles are confined to the blood vessels, the result is an angiographic image of blood vessel morphology. AA is a powerful method for imaging ischemic stroke, as the microbubbles that flow through the circulatory system are not be able to flow through blocked vessels. This allows AA to detect regions of the brain that are not perfused due to ischemic stroke, which indicates the infarct region.
Preclinical ischemic stroke research generally relies on the use of histology and behavioral testing to assess the location and severity of the stroke. Triphenyltetrazolium chloride (TTC) staining is a common histological analysis used to determine the stroke infarct volume. However, it can only be used at an endpoint, since it requires the animal to be euthanized12. Behavioral tests can be used to determine motor function impairment at multiple time points, but they cannot provide quantitative anatomical or physiological values13. Biomedical imaging provides a more quantitative approach to studying the effects of ischemic stroke noninvasively and longitudinally9,14,15. However, existing imaging technologies (such as small-animal magnetic resonance imaging [MRI]) can come at a high cost, be unable to provide concurrent structural and functional information, or have limited penetration depth (as most optical imaging techniques).
Here, we combine photoacoustic, ultrasound, and angiographic tomography (PAUSAT; see system diagram in Figure 1), which allows for complementary structural and functional information of blood perfusion and oxygenation following ischemic stroke16. These are two important aspects in assessing the severity of injury and monitoring the recovery or response to treatments. Using these integrated imaging methods can increase the amount of information obtained by each animal, reducing the number of animals required and providing more information in the study of potential treatments for ischemic stroke.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65319/65319fig01.jpg" /> 
Figure 1: PAUSAT diagram. (A) Complete schematic of the PAUSAT system, including the laser and OPO used for PAT. (B) Inside view of the PAUSAT system, including two ultrasound transducers. The dual-element wobbler transducer is used for both B-mode ultrasound and AA, and the linear-array transducer is used for PAT. Both transducers are mounted on the same 2D motorized stage, allowing for scanning to generate volumetric data. This figure has been modified from16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65319/65319fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>20 GA catheter</td><td>BD Insyte Autoguard Winged</td><td>381534</td><td>For mouse intubation</td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride</td><td>Sigma</td><td>T8877</td><td>Necessary for TTC-staining brain for validation</td></tr><tr><td>532nm Laser</td><td>Quantel</td><td>Q-smart 850</td><td>Laser used to pump the OPO for PAT</td></tr><tr><td>Automatic Ventilator Rovent Jr.</td><td>Kent Scientific</td><td>RV-JR</td><td>To keep mice under anesthesia during surgical procedure</td></tr><tr><td>Black braided silk 4-0 USP</td><td>Surgical Specialties</td><td>SP116</td><td>Used for sutures on the neck for pMCAO surgery</td></tr><tr><td>Bupivacaine</td><td>Hospira</td><td>0409-1159-18</td><td>Used prior to closing wounds during surgical procedure</td></tr><tr><td>C57BL/6 Mice</td><td>Jackson Lab</td><td>#000664</td><td>Mice used for studying ischemic stroke (2-6 month old male/female)</td></tr><tr><td>Clear suture</td><td>Ethicon</td><td>8606</td><td>Used for closing wound (PT stroke and pMCAO). A clear suture won&#039;t interfere with PAT</td></tr><tr><td>Cold Light LED</td><td>Schott</td><td>KL 1600</td><td>Needed to create PT stroke</td></tr><tr><td>Disposable Razor Blade</td><td>Accutec Blades</td><td>74-0002</td><td>For sectioning mouse brain</td></tr><tr><td>Electric drill</td><td>JSDA</td><td>JD-700</td><td>Used to expose MCA during pMCAO procedure</td></tr><tr><td>Electrocauterization tool</td><td>Wet-Field</td><td>Wet-Field Bipolar-RG</td><td>Stops blood flow after drilling during pMCAO procedure</td></tr><tr><td>Hair removal gel</td><td>Veet</td><td>8282651</td><td>Used to remove hair from mouse prior to imaging</td></tr><tr><td>High Temperature Cautery Loop Tip</td><td>BOVIE Medical Corporation</td><td>REF AA03</td><td>Used to avoid bleeding when separating the temporal muscle from the skull</td></tr><tr><td>IR Detector Card</td><td>Thorlabs</td><td>VRC5</td><td>Used to ensure light path is aligned</td></tr><tr><td>Laser Power Meter</td><td>Ophir</td><td>StarBright, P/N 7Z01580</td><td>Can be used to calibrate the laser energy prior to imaging</td></tr><tr><td>Laser Speckle Imaging System</td><td>RWD Life Science Co.</td><td>RFLSI-III</td><td>Can be used to validate stroke surgery success</td></tr><tr><td>Lubricant Eye Ointment</td><td>Soothe</td><td>AB31336</td><td>Can be used to avoid drying of the eyes</td></tr><tr><td>Manually adjustable stage</td><td>Thorlabs</td><td>L490</td><td>Used with custom ramp for multiple focal depth AA imaging</td></tr><tr><td>Modified Vega Imaging System</td><td>Perkin Elmer</td><td>LLA00061</td><td>System containing both B-mode/AA and PAT transducers</td></tr><tr><td>Optical Parametric Oscillator</td><td>Quantel</td><td>versaScan-L532</td><td>Allows for tuning of excitation wavelength in a large range</td></tr><tr><td>Programmable Ultrasound System</td><td>Verasonics</td><td>Vantage 256</td><td>Used for PAT part of system</td></tr><tr><td>Rose Bengal</td><td>Sigma</td><td>330000</td><td>Necessary to induce PT stroke</td></tr><tr><td>Suture</td><td>LOOK</td><td>SP116</td><td>Used for permanent ligation of CCA</td></tr><tr><td>Temperature Contoller</td><td>Physitemp</td><td>TCAT-2</td><td>Used to maintain stable body temperature of mice during procedures</td></tr><tr><td>VesselVue Microbubbles</td><td>Perkin Elmer</td><td>P-4007001</td><td>Used for acoustic angiography (2.43 &amp;times; 10^9 microbubbles/mL)</td></tr></tbody></table>

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.

Imaging of blood vessel morphology in the brain 
AA generates blood vessel morphology images by exciting microbubbles in the circulatory system at their resonant frequency and receiving the super harmonic response of the microbubbles. By using the customized ramp (Figure 2C) attached to a manually adjustable stage, we can image the mouse brain with AA mode at two different focal depths. When deeper regions are targeted, more superficial regions (such as the cerebral cortex) show a poorer resolution and signal strength (Figure 3A), and vice versa (Figure 3B). However, by acquiring two focal depths and combining them, AA images can provide information about a whole coronal section (Figure 3C,D). Additionally, by using the motorized stage to scan along a third dimension, PAUSAT can register a series of coronal images covering the region of interest (ROI) defined by the user. These series of images can be aligned and used to visualize a 3D representation of the whole brain, or the ROI defined by the user. However, additional results demonstrating that other quantitative measures (such as the stroke volume) can be analyzed by using the 3D information from the compilation of images acquired by PAUSAT are also provided here.
Anatomical and functional evaluation before and after ischemic stroke 
To illustrate the potential of the PAUSAT system in preclinical studies, we studied two groups of animals subjected to one of the models of ischemic stroke analyzed here: pMCAO or PT stroke. These two stroke models differ in the principles by which the ischemic region is created. Briefly, in the pMCAO model, the middle cerebral artery is electro-cauterized, stopping the blood supply from this artery to the brain. This injury triggers a secondary injury, in which surrounding tissue becomes ischemic, enlarging the area affected by the stroke. We decided to image the pMCAO stroke brain at day three after the stroke, because this is the time at which the maximum area expected to be affected by the stroke is achieved. In the PT stroke, however, the maximum area of tissue affected by the stroke is achieved after the first day, so we decided to image PT stroke at day one after the stroke was performed. Although we chose these time points in our study, PAUSAT can be used for longitudinal monitoring of stroke at any desired time point.
First, a B-mode scan was acquired to ensure the correct position of the animal&#39;s head and identify the spatial limits between the skull and the brain (Figure 4A and Figure 5A). The most anterior part of the brain imaged includes the frontonasal suture of the skull as a reference point. An ROI of 20 mm (anterior to posterior) x 17.15 mm (lateral) covering the left (contralateral; CL) and the right (ipsilateral; IL) hemispheres was defined in the following studies to locate the stroke region. AA images provided information regarding the blood vessel structures by targeting the signal from the circulating microbubbles. Our results show that in the uninjured brain (baseline), both hemispheres present a similar distribution of blood vessels (Figure 4B and Figure 5B), as expected in the absence of injury. A similar result is observed for the tissue oxygenation map distribution based on PA images obtained at two different wavelengths (Figure 4C and Figure 5C). We decided to evaluate two different wavelengths based on the local maximum optical absorption for deoxygenated hemoglobin (756 nm) and the wavelength at which the deoxygenated and oxygenated hemoglobin presented equal optical absorption (798 nm)20. By capturing these two states of hemoglobin, we can accurately estimate tissue oxygenation (Figure 4D and Figure 5D).
The day after acquiring the baseline image from the brain, we performed surgery on the animal (pMCAO or PT stroke), as described in previous sections. A new set of images were acquired at a specific time point after the stroke (see Figure 2A) to evaluate the location and size of the stroke. The AA images acquired from mice subjected to pMCAO show a notable reduction in the intensity of the signal in the right lateral of the cortex (Figure 4B). The same region shows a reduction in the tissue oxygenation map from PA images, suggesting an ischemic area (Figure 4D). To validate our results, we decided to harvest the brain and perform TTC staining immediately after acquiring the images from the stroke animal. Our results show that the area identified as stroke is similar when compared with our results by PAUSAT and the well-established TTC staining (Figure 4B-E). We also demonstrate here that PAUSAT can identify stroke areas in the brain using a second model of stroke, PT stroke, evaluated at an earlier time point after stroke induction (1 day). As shown in Figure 5B, we were able to identify a region in the upper part of the cortex with a reduced blood flow supply and a concomitant decrease in oxygen saturation (Figure 5D). The results obtained from TTC staining match the location and size of the stroke previously identified by PAUSAT (Figure 5B-E).
The age of the mouse being imaged can heavily affect the image quality. The skull of a mouse becomes thicker with age, and since the skull has a significantly different acoustic impedance relative to soft tissue, a large percentage of the ultrasonic waves are reflected at the skull boundary. Because these integrated imaging systems are all acoustic-based, this leads to a decrease in imaging depth for older mice. This can be clearly seen in Figure 6, where mice of three different ages were imaged with AA under the same conditions. Similar low-signal results are expected if the imaging procedure is not followed as previously described.
Quantitative analysis of the stroke volume by PAUSAT 
As described in the above-mentioned results, PAUSAT captures a series of coronal images targeting an ROI described by the user. We analyzed the volume of stroke by calculating the stroke area within the different coronal images and the distance between images. The stroke volume calculated by PAUSAT shows no statistical difference (p &#60; 0.05) compared to the stroke volume calculated based on a similar approach using TTC staining images (Figure 7).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65319/65319fig03.jpg" /> 
Figure 3: Effect of focal depth of wobbler transducer on the quality of AA images. Images at multiple focal depths can be acquired and later combined to produce the best whole-brain imaging results. (A) AA coronal section acquired at a deeper focal depth. (B) AA coronal section acquired at a more superficial focal depth. (C) Result of fusing deeper focus (green) and more superficially focused (magenta) images. (D) Result of combining deeper focus and more superficially focused images with a pixel scale value comparable to (A) and (B). This figure has been modified from16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65319/65319fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65319/65319fig04.jpg" /> 
Figure 4: Representative PAUSAT results of a mouse brain (#1) before and after pMCAO stroke. (A) Baseline and post-stroke B-mode ultrasound coronal images. (B) Baseline and post-stroke AA coronal images. (C) Baseline and post-stroke PA coronal images at 756 nm excitation. (D) Oxygen saturation map based on 756 nm and 798 nm excitation images. (E) TTC stained sections of a mouse brain, showing the same region of stroke. The distance between the lines on the bottom of the image represents 1 mm. This figure has been modified from16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65319/65319fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65319/65319fig05.jpg" /> 
Figure 5: Representative PAUSAT results of a mouse (#2) before and after PT stroke. (A) Baseline and post-stroke B-mode ultrasound coronal images. (B) Baseline and post-stroke AA coronal images. (C) Baseline and post-stroke PA coronal images at 756 nm excitation. (D) Oxygen saturation map based on 756 nm and 798 nm excitation images. (E) TTC stained sections of a mouse brain, showing the same region of stroke. The distance between the lines on the bottom of the image represents 1 mm. This figure has been modified from16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65319/65319fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65319/65319fig06.jpg" /> 
Figure 6: Effect of mouse age on the quality of AA images. This figure shows the difference in AA signal intensity and imaging depth when animals from different ages are imaged under comparable conditions. As seen in the figure, older mice (18 months) imaging produces lower quality images due to the size and thickness of the skull compared to younger mice (1.5 months), while adult mice show an intermediate signal (6 months). Due to the large acoustic impedance mismatch between the skull and brain tissue, ultrasound waves propagating through the skull are reflected and refracted, leading to signal loss and resolution degradation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65319/65319fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65319/65319fig07.jpg" /> 
Figure 7: Analysis of the volume of the ischemic stroke. (A) Example of TTC sections from PT stroke and corresponding TTC segmented images, AA images, and AA segmented images at comparable coronal locations. (B) The stroke volume calculated based on TTC and AA images for pMCAO and PT stroke, showing no significant differences (p &#62; 0.05). The graph shows the mean &#177; standard deviation (S.D.) (n = 3 animals per group). This figure has been modified from16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65319/65319fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Integrated Photoacoustic, Ultrasound, and Angiographic Tomography PAUSAT for NonInvasive Whole-Brain Imaging of Ischemic Stroke at JoVE.com

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.

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 ...

integrated-photoacoustic-ultrasound-angiographic-tomography-pausat

Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.2K Views.  Duke University. 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.

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

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

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 ...

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 ...

Bioengineering

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

Medicine

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

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.

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 ...

Quantification of Cerebral Perfusion using Laser Speckle Imaging and Infarct Volume using MRI in a Pre-clinical Model of Posterior Circulation Stroke

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical standard to diagnose ischemic stroke include computerized tomography (CT), CT angiography and perfusion and magnetic resonance imaging (MRI). Large animal pre-clinical models to accurately reflect the clinical disease as well as methods to assess stroke burden and evaluate treatments are lacking.
Methods: We describe a canine model of large vessel occlusion (LVO) stroke in the posterior circulation, and developed a laser speckle imaging (LSI) protocol to monitor perfusion changes in real time.&#160; We then utilized high b-value DWI (b=1800s/mm2) MRI to increase detection sensitivity. We also evaluated the ability of magnetic resonance angiography (MRA) to assess arterial occlusion and correlate with DSA. Finally, we verified infarct size from apparent diffusion coefficient (ADC) mapping with histology.&#160;
Results:&#160; Administration of thromboembolism occluded the basilar artery as tracked by DSA (n=7).&#160; &#160;LSI correlated with DSA, demonstrating a reduction in perfusion after stroke onset that persisted throughout the experiment, allowing us to monitor perfusion in real time.&#160; DWI with an optimized b-value for dogs illustrated the stroke volume and allowed us to derive ADC and magnetic resonance angiography (MRA) images. The MRA performed at the end of the experiment correlated with DSA performed after occlusion. Finally, stroke burden on MRI correlated with histology.
Conclusions: Our studies demonstrate real time perfusion imaging using LSI of a canine thromboembolic LVO model of posterior circulation stroke, which utilizes multimodal imaging important in the diagnosis and treatment of ischemic stroke.

A canine model of LVO stroke was utilized to develop laser speckle imaging to monitor cerebral perfusion in real-time.&#160; Diffusion-weighted MRI was optimized to image infarct volume utilizing a high b-value, enabling ADC and MRA, correlated with DSA at the time of stroke.&#160; Finally, ADC reconstructions correlated with histological findings.

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical ...

Photoacoustic Tomography to Image Blood and Lipids in the Infrarenal Aorta

Source: Gurneet S. Sangha and <a href="https://www-jove-com.remotexs.ntu.edu.sg/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
Photoacoustic tomography (PAT) is an emerging biomedical imaging modality that utilizes light generated acoustic waves to obtain compositional information from tissue. PAT can be used to image blood and lipid components, which is useful for a wide variety of applications, including cardiovascular and tumor imaging. Currently used imaging techniques have inherent limitations that restrict their use with researchers and physicians. For example, long acquisition times, high costs, use of harmful contrast, and minimal to high invasiveness are all factors that limit the use of various modalities in the laboratory and clinic. Currently, the only comparable imaging techniques to PAT are emerging optical techniques. But these also have disadvantages, such as limited depth of penetration and the need for exogenous contrast agents. PAT provides meaningful information in a rapid, noninvasive, label-free manner. When coupled with ultrasound, PAT can be used to obtain structural, hemodynamic, and compositional information from tissue, thereby complementing currently used imaging techniques. The advantages of PAT illustrate its capabilities to make an impact in both the preclinical and clinical environment.

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

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Photoacoustic Tomography to Image Blood and Lipids in the Infrarenal Aorta