Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature
May 30th, 2019
A compact pulsed laser diode-based desktop photoacoustic tomography (PLD-PAT) system is demonstrated for high-speed dynamic in vivo imaging of small animal cortical vasculature.
Photoacoustic tomography, or PAT, is an imaging hybrid biomedical imaging modality, combining both optical and ultrasound imaging for monitoring dynamic changes in small animal brains. This is a cost-effective, portable high speed imaging system that uses imaging speed as short as half a second. Demonstrating the procedure with Sandeep will be Praveen, a PhD student, Dr.Paul, a research fellow, and Rhonnie, a research assistant.
Begin by mounting all single element ultrasound transducers onto each transducer holder one at a time, such that the surface of each acoustic reflector faces toward the center of the scanning area. Use connecting cables to connect each transducer cable to the low noise signal amplifier. Switch on the power supply of the chiller and turn on the chiller switch to set the temperature between 20 to 25 degrees Celsius.
Switch on the low voltage power supply and slowly turn the current control to 0.3 amps. Set the voltage to 12 volts and verify that the current does not exceed 0.1 amp. Switch on the high voltage power supply and press the preset button to set the current to one amp and the voltage to zero volts.
Enable the output button and switch on the power supply of the function generator. Press the recall button and select a two kilohertz configuration to generate the laser pulses at this repetition rate. Place the acrylic tank inside the scanner and fill the tank with water until the detecting surfaces of the transducers are completely immersed.
Then switch on the power supply of the low noise signal amplifier. Place the anesthetized rat in the prone position on the workbench. After confirming a lack of response to toe pinch, use a hair trimmer to remove the fur on the scalp.
Gently apply depilatory cream to remove the fur completely from the exposed skin using a cotton swab to remove the cream after four to five minutes. Apply ointment to animal's eyes and mount a custom made animal holder equipped with a breathing mask on a lab jack. Place the rat in the prone position on the holder with the head resting on the horizontal platform of the holder.
Use surgical tape to secure the animal to the holder. Clamp the pulse oximeter to one hind limb to monitor its physiological condition. Use a cotton tipped applicator to apply a layer of colorless ultrasound gel to the scalp.
Then adjust the lab jack position to the center of the scanner. Adjust the height of the jack manually so that the imaging plane is at the center of the acoustic reflector. After setting the parameters in the data acquisition software for a 360 degree acquisition scan, enable the output of the function generator to turn on the pulsed laser diode laser emission.
Slowly increase the voltage of the variable high voltage power supply to 120 volts for maximum per pulse energy. Run the data acquisition software program to rotate all eight of the transducers 360 degrees over a four second scan time. Disable the output of the function generator to turn off the laser emission.
Using the reconstruction algorithm in the data processing software, determine the scanning radius of all eight of the transducers by trial and error, using the back-projection algorithm. Set the parameters in the data acquisition software for a 45 degree acquisition over a 0.5 second scan time. Enable the output of the function generator to turn on the laser emission.
Run the data acquisition software program to rotate all eight of the transducers 45 degrees to obtain the initial control data. Disable the output of the function generator to turn off the laser emission. Next, inject 0.3 milliliters of indocyanine green into the tail vein of the rat.
Enable the output of the function generator to turn on the laser emission. Then run the data acquisition software to acquire A-lines over a 0.5 second scan time in a 45 degree rotation. At the end of the acquisition, use the back-projection algorithm to reconstruct the cross sectional brain image from the saved A-lines.
Here, brain images from a 98 gram female rat, collected at four and 0.5 second scan speeds are shown. The sagittal sinus and the transfer sinus are clearly visible in both images. The photographs show the same region of the rat brain before and after removing the scalp.
Photoacoustic tomography imaging can achieve the same visualization noninvasively with the skin and skull intact. In this plot, increases in the average photoacoustic signal in the sagittal sinus due to increases and subsequently decreases in the optical absorption by indocyanine green at 860 nanometer wavelengths over time can be observed. Always take extra care when injecting the ICG dye into the tail vein of the animal.
This low cost desktop photoacoustic tomography system can be used for the dynamic imaging of small animal brain vasculatures with high special resolution at 0.5 seconds scan speed. Other applications of this imaging system include brain tumor imaging, the imaging of different organs in small animals and other therapeutic applications.
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Overview
This article presents a compact pulsed laser diode-based desktop photoacoustic tomography (PLD-PAT) system designed for high-speed dynamic in vivo imaging of small animal cortical vasculature. The system combines optical and ultrasound imaging modalities to monitor changes in small animal brains effectively.
Key Study Components
Area of Science
- Biomedical Imaging
- Neuroscience
- Photoacoustic Tomography
Background
- Photoacoustic tomography (PAT) is a hybrid imaging technique.
- It integrates optical and ultrasound imaging for enhanced monitoring.
- The system is portable and cost-effective.
- Imaging speeds can be as short as half a second.
Purpose of Study
- To demonstrate a new imaging system for small animal studies.
- To improve the monitoring of dynamic changes in cortical vasculature.
- To provide a practical solution for in vivo imaging.
Methods Used
- Mounting ultrasound transducers onto holders.
- Connecting transducer cables to a low noise signal amplifier.
- Using a chiller to maintain optimal temperature conditions.
- Conducting imaging procedures with trained personnel.
Main Results
- The PLD-PAT system successfully captured dynamic imaging data.
- High-speed imaging was achieved with minimal setup time.
- The system demonstrated effectiveness in visualizing cortical vasculature.
- Results indicate potential for broader applications in neuroscience.
Conclusions
- The PLD-PAT system is a promising tool for in vivo imaging.
- It offers advantages in speed and cost for research applications.
- Future studies may expand its use in various biomedical fields.
Frequently Asked Questions
Executive Industry Relevance
This compact pulsed laser diode-based photoacoustic tomography system enables high-speed dynamic imaging of cortical vasculature in small animals, supporting early-stage target validation and mechanistic de-risking in neuroscience drug discovery. By providing quantitative, real-time visualization of dye wash-in and wash-out processes, the system enhances predictive confidence in hemodynamic biomarker assessment and reduces biological ambiguity in preclinical models. Its portability, cost-effectiveness, and sub-second imaging speed position it as a scalable tool for integration into discovery workflows focused on neurovascular targets and blood-brain barrier penetration studies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses related to neurovascular function and blood-brain barrier permeability through direct visualization of contrast agent dynamics.
- Operational Value: Supports biological de-risking by providing reproducible, quantitative hemodynamic readouts that clarify target engagement and pathway modulation in vivo.
- Predictive Value: Facilitates portfolio triage by generating early, translatable biomarkers of vascular response that correlate with drug-induced physiological changes.
Screening & Assay Development
- Assay Readiness: Prepares validated biological systems for downstream screening by establishing baseline vascular function and dye clearance kinetics in disease-relevant cortical models.
- Quantitative Output: Delivers standardized, high-temporal-resolution photoacoustic signal measurements that enable reliable comparison of compound effects on cerebral blood flow and volume.
- Platform Scalability: The desktop PLD-PAT system’s compact design and rapid setup support reuse across multiple studies, enhancing assay standardization and cross-lab reproducibility.
Translational & Preclinical Research
- Translational Continuity: Bridges discovery and preclinical validation by enabling longitudinal monitoring of vascular responses in the same animal model, reducing variability and improving data confidence.
- Mechanistic De-risking: Provides direct, non-invasive evidence of target-mediated vascular effects, supporting go/no-go decisions before investing in larger-scale pharmacokinetic or toxicology studies.
- Disease Model Alignment: Applicable to rodent models of stroke, neurodegeneration, and brain tumors where cortical vasculature integrity is a key pathophysiological feature and therapeutic target.
Pipeline & Workflow Integration
The PLD-PAT system fits within the early discovery to preclinical continuum, supporting hypothesis testing in target validation, assay readiness in screening, and quantitative hemodynamic analytics in translational research, with potential for enterprise reuse across neurovascular discovery programs.
- Discovery Biology: Supports mechanistic hypothesis testing by enabling real-time, non-invasive visualization of cortical vascular responses to pharmacological perturbations.
- Screening: Enhances assay readiness through reproducible baseline vascular imaging and dynamic contrast agent tracking, critical for evaluating vasoactive compounds.
- Analytics: Generates quantitative, time-resolved photoacoustic signal measurements in regions of interest (e.g., sagittal sinus) that allow objective comparison of hemodynamic changes across experimental conditions.
- Translational Research: Connects early vascular phenotypes to preclinical outcomes by enabling longitudinal, within-subject monitoring of dye uptake and clearance, improving risk-adjusted advancement decisions.
- Enterprise Reuse: The system’s standardized protocol, low operational cost, and rapid scan speed support deployment as a shared core capability across multiple discovery teams and projects.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity through direct, quantitative observation of neurovascular function.
- Operational Value: Delivers standardization and reproducibility via fixed-geometry transducer arrays, controlled laser pulse repetition, and automated reconstruction algorithms.
- Strategic Value: Improves capital efficiency by enabling high-frequency, longitudinal imaging in small animals, reducing the number of subjects needed for robust statistical inference.
- Portfolio Impact: Supports risk-adjusted prioritization by providing early, translatable vascular biomarkers that inform go/no-go decisions in neurovascular and CNS drug programs.
Implementation Considerations
- Requires expertise in small animal handling, surgical preparation, and photoacoustic image reconstruction.
- Depends on synchronized operation of pulsed laser diode, function generator, multi-channel transducer array, and data acquisition software.
- Necessitates cross-team standardization of anesthesia protocols, dye dosing, and region-of-interest selection for consistent hemodynamic measurements.
- Adaptation to other model systems (e.g., mice, larger rodents) may require adjustments to transducer frequency, laser wavelength, and scanning geometry.
- Practical limitations include dependence on optimal optical absorption contrast (e.g., ICG at 860 nm) and potential signal attenuation in deeper cortical layers or through intact skull in larger animals.
Biopharma FAQs
Chapters in this video
0:04
Title
0:43
Pulsed Laser Diode (PLD)-Based Desktop Photoacoustic Tomography (PAT) System Preparation
2:10
Imaging Preparation
3:24
Dynamic In Vivo Indocyanine Green (ICG) Uptake and Clearance
5:15
Results: Representative Non-Invasive In Vivo Desktop PLD-PAT Imaging and ICG Pharmacokinetics
6:08
Conclusion
