The research is supported by the Singapore Ministry of Health&#8217;s National Medical Research Council (NMRC/OFIRG/0005/2016: M4062012). The authors would like to thank Mr. Chow Wai Hoong Bobby for the machine shop support.

All animal experiments were performed according to the guidelines and regulations approved by the Institutional Animal Care and Use Committee of Nanyang Technological University, Singapore (Animal Protocol Number ARF-SBS/NIE-A0331).
1. System description
<ol>
	<li>Mount the PLD laser into the circular scanner and mount the optical diffuser (OD) in front of the PLD exit window to make the output beam homogeneous, as shown in Figure 1A. Connect the PLD to the laser driver unit (LDU). 
	NOTE: The PLD generates ~816 nm wavelength pulses, pulses of ~107 ns in duration, and up to a 2 KHz repetition rate with a maximum pulse energy of ~3.4 mJ. The LDU consists of chiller, 12 V power supply, variable high voltage power supply to control the laser power, and function generator to change the pulse repetition rate.</li>
	<li>Mount all eight SUTRs on each SUTR holder one-by-one such that the surface of each acoustic reflector faces towards the center of the scanning area, as shown in Figure 1B. Connect each SUTR cable to the low-noise signal amplifier with the help of connecting cables. 
	NOTE: The central frequency of the ultrasound transducer is 5 MHz and has a 13 mm diameter active area. Two amplifiers each of 24 dB gain are connected in series for each channel.</li>
	<li>Switch on the power supply of the chiller, then turn on the switch of the chiller to set the temperature between 20 &#176;C and 25 &#176;C.</li>
	<li>Switch on the supply of the low voltage power supply and slowly turn the current control to set the current limit at 0.3 A. Set the voltage to 12 V. Verify that the current does not exceed 0.1 A.</li>
	<li>Switch on the supply of the high voltage power supply. Press the &#8220;Preset&#8221; button and set the current to 1 A and voltage to 0 V. Enable the &#8220;Output&#8221; button: 0 V/0 A.</li>
	<li>Switch on the power supply of the function generator. Press the &#8220;Recall&#8221; button and choose a 2 KHz configuration to generate the laser pulses at this repetition rate.</li>
	<li>Place acrylic tank inside the scanner as shown in Figure 1A and fill the tank with water such that the detecting surface of the SUTRs are immersed completely inside water.</li>
	<li>Make sure all the SUTRs detecting surfaces are inside the water medium. Switch on the power supply of the low-noise-signal amplifier.</li>
</ol>
2. Animal preparation for rat brain imaging
NOTE: Healthy female rats (see Table of Materials) were used to demonstrate the above described desktop PLD-PAT system for imaging small animal cortical vasculature.
<ol>
	<li>Hold the animal on its back by arresting the head and body motion. Anesthetize the animal by intraperitoneal injection of a mixture of 2 mL of ketamine (100 mg/mL), 2 mL of xylazine (20 mg/mL), and 1 mL of saline (dosage of 0.2 mL/100 g). 
	NOTE: After the injection, the animal&#8217;s toe is pinched to test for any positive reflexes such as leg or body movements, vocalization, or marked increases in respirations. An absence of such reflex actions confirms successful anesthetization of the animal.</li>
	<li>To prevent dryness due to anesthesia and laser illumination, very carefully apply artificial tear ointment to the rat eyes. Place the animal in prone position on the working bench and remove the fur on the scalp of the animal using a hair trimmer and gently apply hair removal cream to the shaved area and remove the fur completely.
	<ol>
		<li>After 4&#8211;5 min, remove the applied cream using a cotton swab.</li>
	</ol>
	</li>
	<li>Mount the custom-made animal holder (see Table of Materials) equipped with a breathing mask (see Table of Materials) on a lab-jack.</li>
	<li>Place the animal in prone position on the holder so that the head rests on the horizontal platform of the holder. Use surgical tape to secure the animal to the holder.</li>
	<li>Ensure that the breathing mask covers the nose and mouth of the rat to deliver anesthesia mixture. The breathing mask is customized to suit the imaging window. 10% of the commercially available nose cone is cut and then connected to a piece of glove.</li>
	<li>Connect the breathing mask to the anesthesia machine before switching it on.</li>
	<li>Switch on the anesthesia machine and set it to deliver anesthetic mixture containing 1.0 L/min of oxygen with 0.75% isoflurane to the animal breathing mask.
	<ol>
		<li>Clamp the pulse oximeter to one of the animal&#8217;s hind legs to monitor its physiological condition.</li>
	</ol>
	</li>
	<li>Apply a layer of colorless ultrasound gel to the scalp of the rat using a cotton tipped applicator. Adjust the lab-jack position to the center of the scanner and adjust the height of the lab-jack manually so that the imaging plane is at the center of the acoustic reflector.</li>
</ol>
3. Dynamic in vivo imaging of uptake and clearance process of ICG in rat brain
<ol>
	<li>Set the parameters in the data acquisition software (see Table of Materials) for a 360&#176; acquisition scan.</li>
	<li>Turn on the PLD laser emission by enabling the output of the function generator (laser emission will start). Then, slowly increase the voltage of the variable high voltage power supply to 120 V for maximum per pulse energy.</li>
	<li>Run the data acquisition software (see the Table of Materials) program to rotate all eight SUTRs in 360&#176; over a 4 s scan time. 
	NOTE: For example, if the SUTRs are rotated for 4s, the PLD delivers 8,000 (= 4 x 2,000) pulses and each SUTR collects 8000 A-lines. These 8,000 A-lines are reduced to 400 by averaging over 20 signals (after averaging A-lines = 8,000/20 = 400). A reconstruction program based on delay-and-sum back projection algorithm is used to find out the scanning radius of each SUTR.</li>
	<li>Disable the output of the function generator to turn off the laser emission.</li>
	<li>Using the reconstruction algorithm in data processing software (see Table of Materials) find out the scanning radius of all eight SUTRs by trial-and-error, using the back-projection algorithm.</li>
	<li>Set the parameters in the data acquisition software (see Table of Materials) for 45&#176; acquisition over a 0.5 s scan time. 
	NOTE: For example, if the SUTRs are rotated for 0.5s, the PLD delivers 1,000 (= 0.5 x 2,000) pulses and each SUTR collects 1000 A-lines. These 1,000 A-lines are reduced to 400 by averaging over 20 signals (after averaging A-lines = 1,000/20 = 50).</li>
	<li>Enable the output of the function generator to turn on the laser emission.</li>
	<li>Run the data acquisition software (see Table of Materials) program to rotate all eight SUTRs in 45&#176; to obtain initial control data before administering ICG.</li>
	<li>Disable the output of the function generator to turn off the laser emission.</li>
	<li>Identify the tail vein of the animal and inject 0.3 mL of ICG (see Table of Materials) (323 &#956;M) into the tail vein of the rat.</li>
</ol>
4.
NOTE: 1.25 mg of ICG powder was weighed using a micro-weighing machine and mixed with 5 mL of distilled water to obtain a concentration of 323 &#956;M for the ICG solution.
<ol>
	<li>Enable the output of the function generator to turn on the laser emission.</li>
	<li>Run the data acquisition software (see Table of Materials) program to acquire A-lines over a 0.5 s scan time in 45&#176; rotation.</li>
</ol>
5.
NOTE: A-lines acquired during a 0.5 s scan time are used to generate one cross-sectional image. There is time gap of ~0.4&#8211;0.6 s between each scan.
<ol>
	<li>After the data acquisition is over, using the back-projection algorithm in data processing software (see Table of Materials), reconstruct the cross-sectional brain image from the saved A-lines.</li>
	<li>Turn off the laser and then turn off anesthesia machine, lower the lab-jack and remove the animal from the stage. Return the animal to the cage and monitor until it regains consciousness.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59764/59764fig1.jpg" /> 
Figure 1: Schematic of the desktop PLD-PAT system. (A) Schematic of the desktop PLD-PAT set up. PLD: pulsed laser diode, OD: optical diffuser, SUTR: acoustic reflector based single-element ultrasound transducer, AM: anesthesia machine, CSP: circular scanning plate, SM: stepper motor, LDU: laser driving unit, AMP: amplifier, DAQ: data acquisition card. (B) Circular arrangement of eight SUTRs around the scanning center. <a href="https://www.jove.com/files/ftp_upload/59764/59764fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have no relevant financial interests or potential conflicts of interest to disclose.

This work presents a protocol to use a desktop PLD-PAT system for conducting experiments on small animals like rats for in vivo brain imaging and dynamic fast-uptake and clearance process of contrast agents like ICG. Bulky, expensive OPO-PAT systems take several minutes (2-5 min) to acquire a single cross-sectional in vivo image. A compact, low-cost, first generation portable PLD-PAT system provides single cross-sectional in vivo images in 5 s. In contrast, a high-speed, compact, low-cost desktop PLD-PAT system renders a...

Photoacoustic computed tomography (PACT/PAT) is a promising non-invasive biomedical imaging modality combining rich&#160;optical contrast with high&#160;ultrasond&#160;resolution1,2,3,4,5. When a nanosecond pulsed laser deposits energy onto light absorbing chromophores&#160;present inside any biological tissue, local temperature increases leading to thermoelastic expansion and contraction of the tissue, resulting in generation of pressure waves. These pressure waves are known as ultrasound waves or photoacoustic (PA) waves, which can be detected by ultrasound transducers around the sample. The detected PA signals are reconstructed using various reconstruction algorithms6,7,8,9 to generate cross-sectional PA images. PA imaging provides structural and functional information from macroscopic organs to microscopic organelles due to the wavelength dependence of endogenous chromophores present inside the body10. PAT imaging has been successfully used for breast cancer detection1, sentinel lymph node imaging11, mapping of oxyhemoglobin (HbO2), deoxyhemoglobin (HbR), total hemoglobin concentration (HbT), oxygen saturation (SO2)12,13, tumor angiogenesis14, small animal whole body imaging15, and other applications.
Nd:YAG/OPO lasers are conventional excitation sources for first generation PAT systems that are widely used in photoacoustic community for small animal imaging and deep tissue imaging16. These lasers provide ~100 mJ energy pulses at low repetition rates of ~10-100 Hz. The PAT imaging systems using these costly and bulky lasers are not suitable for high-speed imaging with single-element ultrasound transducers (SUTs), due to the limited pulse repetition rate. This inhibits real-time monitoring of physiological changes occurring at high speeds inside the animal. Using array-based transducers like linear, semi-circular, circular, and volumetric arrays with Nd:YAG laser excitation, high-speed imaging is possible. However, these array transducers are expensive and provide lower sensitivities compared to SUTs; yet, the imaging speed is limited by the low repetition rate of the laser. State-of-the-art single-impulse PACT systems with customized full-ring array transducer obtain the PA data at 50 Hz frame rates17. These array transducers need complex back-end receiving electronics and signal amplifiers, making the overall system more expensive and difficult for clinical use.
Their compact size, lower cost requirements, and higher pulse repetition rate (order of KHz) make pulsed laser diodes (PLDs) more promising for real-time imaging. Due to these advantages, PLDs are actively used as an alternate excitation source in second generation PAT systems. PLD-based PAT systems have been demonstrated successfully for high-frame rate imaging using array transducers18, deep-tissue and brain imaging19,20,21, cardiovascular disease diagnosis22, and rheumatology diagnosis23. As SUTs are highly sensitive and less expensive compared to array transducers, they are still extensively used for PAT imaging. Fiber-based PLD system have been demonstrated for phantom imaging24. A portable PLD-PAT system has been demonstrated previously by mounting the PLD inside the PAT scanner25. With one SUT circular scanner, phantom imaging was performed during 3 s of scan time, and in vivo rat brain imaging was performed during a 5 s period using this PLD-PAT system19.
Furthermore, improvements have been made to this PLD-PAT system to make it more compact and create a desktop model using eight acoustic reflector-based single-element ultrasound transducers (SUTRs)26,27. Here, SUTs were placed in a vertical instead of horizontal direction with the aid of a 90&#176; acoustic reflector28. This system can be employed for scan times of up to 0.5 s and ~3 cm deep in tissue imaging and in vivo small animal brain imaging. In this work, this desktop PLD-PAT system is used to provide the visual demonstration of experiments for in vivo brain imaging in small animals and for dynamic visualization of uptake and clearance process of Food and Drug Administration (FDA)-approved indocyanine green (ICG) dye in rat brains.

<table><tbody><tr><td>12 V power supply</td><td>Voltcraft</td><td>PPS-11810</td><td>To supply operating voltage for PLD</td></tr><tr><td>Acoustic reflector</td><td>Olympus</td><td>F102</td><td>45 degree reflector augmented to the ultrasound transducer</td></tr><tr><td>Acrylic water tank</td><td>NTU workshop</td><td>Custom-made</td><td>It is used to hold water that acts as an acoustic coupling medium between animal brain and detector</td></tr><tr><td>Anesthetic Machine</td><td>Medical plus pte ltd</td><td>Non-Rebreathing Anaesthesia machine with oxygen concentrator.</td><td>Supplies oxygen and isoflurane to animal</td></tr><tr><td>Animal distributor</td><td>In Vivos Pte Ltd, Singapore</td><td></td><td>Animal distributor that supplies small animals for research purpose</td></tr><tr><td>Animal holder</td><td>NTU workshop</td><td>Custom-made</td><td>Used for holding animal on its abdomen</td></tr><tr><td>Breathing mask</td><td>NTU workshop</td><td>Custom-made</td><td>Used along with animal holder to supply anesthesia mixture to the animal</td></tr><tr><td>Circular Scanner</td><td>NTU workshop</td><td>Custom-made</td><td>Scanner is made out of aluminum</td></tr><tr><td>DAQ (Data acquisition) Card</td><td>Spectrum</td><td>M2i.4932-exp</td><td>16 bit, 30 Ms/s, 8 channels, 1 Gs, PCIe</td></tr><tr><td>Data acqusition software</td><td>National Instruments Corporation,Austin,TX,USA)</td><td>NI LabVIEW 2015 SP1 (32 bit)</td><td>LabVIEW based program developed in our laboratory for controlling the stepper motor and acquring the PA singnals from the detector</td></tr><tr><td>Data processing software</td><td>Matlab (Mathworks, Natick, MA, USA)</td><td>Matlab R2015b</td><td>Matlab code developed in our laboratory for reconstructing cross-sectional PA images</td></tr><tr><td>Function generator</td><td>RIGOL</td><td>DG1022</td><td>To change the repetition rate of the PLD. It will provide TTL signal to synchronize the DAQ with the laser excitation.</td></tr><tr><td>Low noise signal amplifier</td><td>Genetron</td><td>Custom-made using Mini-circuits, ZFL-500LN-BNC</td><td>To receive, and amplify the PA signal from SUTR. Its gain is 24 dB.</td></tr><tr><td>Optical diffuser</td><td>Thorlabs</td><td>DG-120</td><td>Used to to make the laser beam homogeneous</td></tr><tr><td>Pulsed laser diode</td><td>Quantel, France</td><td>QD-Q1924-ILO-WATER</td><td>It is the excitation laser source with specifications of 816 nm wavelength, 3.4 mJ per pulse energy, 107 ns pulse width, 2 KHz maximum pulse repitition rate, dimensions : 13.0 x 7.6 x 5.0 cm</td></tr><tr><td>Rats</td><td>In Vivos Pte Ltd, Singapore</td><td>NTac:SD, Sprague Dawley / SD</td><td>Female, weight 100&amp;plusmn;10g, strain of rats: Sprague Dawley, age: 4-5 weeks</td></tr><tr><td>Stepper motor with gearbox</td><td>LIN Engineering (Servo Dynamics)</td><td>Motor: CO-5718L-01P-RO, Gearbox: DPL64/1; Power supply PW-100-24</td><td>To move the detector holder in a circular geometry. Torque: 2.08 N-m, Rotor inertia: 2.6 kg-cm2</td></tr><tr><td>Ultrasound gel</td><td>Progress/parker acquasonic gel</td><td>PA-GEL-CLEA-5000</td><td>Clear ultrasound gel</td></tr><tr><td>Ultrasound Transducer</td><td>Olympus</td><td>V309-SU/ U8423013</td><td>Ultrasonic sensors used for photoacoustic detection. Central freqency 5 MHz, 0.5 in</td></tr><tr><td>Variable high voltage power supply</td><td>Elektro-Automatik</td><td>EA-PS 8160-04 T</td><td>To change the laser output power</td></tr></tbody></table>

pulsed laser diode-based desktop photoacoustic tomography for monitoring wash-in and wash-out of dye in rat cortical vasculature

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. Custom-made circular ring array-based transducers and conventional bulky Nd:YAG/OPO lasers inhibit translation of the PAT system to clinics. Ultra-compact pulsed laser diodes (PLDs) are currently being used as an alternative source of near-infrared excitation for PA imaging. High-speed dynamic in vivo imaging has been demonstrated using a compact PLD-based desktop PAT system (PLD-PAT). A visualized experimental protocol using the desktop PLD-PAT system is provided in this work for dynamic in vivo brain imaging. The protocol describes the desktop PLD-PAT system configuration, preparation of animal for brain vascular imaging, and procedure for dynamic visualization of indocyanine green (ICG) dye uptake and clearance process in rat cortical vasculature.&#160;

The potentiality of the described desktop PLD-PAT system for dynamic in vivo brain imaging has been showcased in this protocol with corresponding results. High-speed imaging capability of the desktop PLD-PAT system was demonstrated by performing in vivo brain imaging of healthy female rats. PA signals were collected using eight SUTRs rotating in 360&#176; and 45&#176; around the rat brain at scan speeds of 4 s and 0.5 s, respectively. Figure 2A,B show brain images of a fe...

Watch this Scientific Journal Video about Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature at JoVE.com

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

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 (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. ...

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Research

JoVE Journal

Bioengineering

7.9K Views.  Nanyang Technological University. 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.

Video: Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

Three-dimensional Optical-resolution Photoacoustic Microscopy

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

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

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

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

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

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

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

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

Biology

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory alterations of the spinal cord. In this article, our goal was to use both Laser Doppler imaging and monitoring to analyze the change of BF after spinal cord injury. Both the laser Doppler image scanner and the probe/monitor were being employed to obtain each readout. The data of LDPI provided a local distribution of BF, which gave an overview of perfusion around the injury site and made it accessible for comparative analysis of BF among different locations. By intensely measuring the probing area over a period of time, a combined probe was used to simultaneously measure the BF and oxygen saturation of the spinal cord, showing overall spinal cord perfusion and oxygen supply. LDF itself has a few limitations, such as relative flux, sensitivity to movement, and biological zero signal. However, the technology has been applied in clinical and experimental study due to its simple setup and rapid measurement of BF.

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on rat.

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory ...

Behavior

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

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

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

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

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

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

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

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

Neuroscience

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

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

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

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

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 ...

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

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

Summary

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Chapters in this video

Three-dimensional Optical-resolution Photoacoustic Microscopy

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

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

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

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

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

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

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo