This work was supported by an Innovative Engineering for Health award by the Wellcome Trust (No. WT101957) and the Engineering and Physical Sciences Research Council (EPSRC) (No. NS/A000027/1), by a Wellcome/EPSRC Centre award [203145Z/16/Z &#38; NS/A000050/1], by a Starting Grant from the European Research Council (Grant No. ERC-2012-StG, Proposal 310970 MOPHIM), and by an EPSRC First Grant (No. EP/J010952/1). A.L.D. is supported by the UCL/UCLH NIHR Comprehensive Biomedical Research Centre. The authors are grateful to the staff of the Royal Veterinary College for their valuable assistance with in vivo experiments.

1. System Hardware
<ol>
	<li>The clinical custom ultrasound imaging probe
	<ol>
		<li>Create a draft design for the layout of the transducer elements in the custom probe that includes central and side arrays. Submit the design to the manufacturer of this probe.</li>
		<li>With feedback from the manufacturer, create a detailed design for the custom probe that includes refinements to the transducer frequency characteristics and geometries (Figure 2). 
		NOTE: Typically, the manufacturer of the custom probe can design the electronic systems, the probe housing, and the probe connector for compatibility for a particular type of ultrasound imaging system. The manufacturer can also include an operation mode switch (hardware) to determine which set of 128 elements was addressed by the ultrasound imaging system. In imaging mode, the central array is addressed; in tracking mode, the side arrays are addressed.</li>
	</ol>
	</li>
	<li>The tracking needle
	<ol>
		<li>Select a fiber-optic ultrasound hydrophone which comprises a single-mode optical fiber with a Fabry-P&#233;rot cavity at the distal end (outer diameter (OD): 150 &#181;m). 
		NOTE: Hydrophones that comprise a single-mode optical fiber with a Fabry-P&#233;rot cavity at the distal end (OD: 150 &#181;m), are available commercially. Proximal to the distal end, optical fibers that are frequently used for telecommunications have a cladding layer (OD: 125 &#181;m), a buffer layer (OD: 250 &#181;m), and a jacket (OD: 900 &#181;m).</li>
		<li>Using a scalpel, partially remove the 900 micrometer jacket along the length of the fiber optic hydrophone, close to its distal end, to expose the buffer layer until the hydrophone can fit within the needle cannula. 
		NOTE: For mechanical robustness, it is useful to retain the protective buffer layer/jacket on the section of the fiber optic cable that is proximal to the Luer connector. Take care with handling the fragile section of the fiber after the jacket is removed, before it is protected by the needle cannula.</li>
		<li>Affix the medical needle horizontally to a manual horizontal translation stage, and visualize the needle tip with a stereo microscope, with the optical axis of the microscope aligned horizontally and perpendicular to the needle. If necessary, rotate the needle about its axis so that the bevel surface of the needle can be seen with the microscope.</li>
		<li>With the distal end of the needle in view of the microscope, insert the fiber-optic ultrasound receiver through the cannula of a Tuohy-Borst Sidearm adapter and subsequently through the Luer connector of the needle until the sensing region of the hydrophone is just proximal to the bevel surface of the needle. At this stage, the Sidearm adapter should not be connected to the needle. Affix the hydrophone to the translation stage (polyimide tape works well) to avoid its movement within the needle.</li>
		<li>Affix the hydrophone to the translation stage with polyimide tape to avoid movement of the device within the needle.</li>
		<li>Vertically affix a 20-microliter pipette to the vertical translation stage with the tip facing downward and use both the horizontal and vertical translation stages to position the micropipette tip until it is adjacent to the fiber-optic hydrophone and about 0.5 mm proximal to the sensing region at the distal end.</li>
		<li>Place a drop of optical adhesive at the proximal end of the micropipette and adjust the needle to allow a direct path from the micropipette tip to the fiber-optic ultrasound receiver.</li>
		<li>Then use a 10-mL syringe to apply pressure at the proximal end of the micropipette to gradually dispense the adhesive from the distal into the fiber-optic ultrasound receiver, taking care to avoid applying adhesive to the sensing region or occluding the cannula, and illuminate the needle tip with ultraviolet light until the optical adhesive is cured.</li>
	</ol>
	</li>
</ol>
2. System Integration
<ol>
	<li>Connect the hydrophone to its optical console. 
	NOTE: Optical consoles that provide an analog voltage signal proportional to the received pressure are available commercially.</li>
	<li>Connect the custom ultrasound imaging probe to the ultrasound console.</li>
	<li>Perform interleaved acquisitions of B-mode ultrasound images and coded ultrasound pulses for tracking10,14. For B-mode ultrasound image acquisition, perform pulse-echo transmit-receive sequences with the central array elements. Use the hardware switch to control whether the side array elements or the central array elements are accessed.</li>
	<li>Digitize the hydrophone signals and the timing signals according to the starts of ultrasound transmissions simultaneously with a data acquisition (DAQ) card.</li>
	<li>Process and display the signals acquired from the pulse-echo transmit-receive sequences, to obtain B-mode ultrasound images. Additionally, process and display the hydrophone signals to localize the fiber-optic ultrasound receiver relative to the custom probe. For the latter task, the algorithms are described by Xia et al.12,14</li>
	<li>Overlay the needle tip locations onto the B-mode ultrasound images. To display 3D tracking information onto a 2D ultrasound image display, the position of the needle tip (lateral and depth coordinates) can be indicated with a cross; the out-of-plane distance and side of the imaging plane, with the size and color of this cross, respectively.</li>
</ol>
3. Pre-clinical Validation
<ol>
	<li>Select the operation mode using the switch on the ultrasound imaging probe.</li>
	<li>Add ultrasound gel to the custom ultrasound imaging probe.</li>
	<li>Prepare a fetal ultrasound phantom by adding water to mimic amniotic fluid.</li>
	<li>Using the B-mode ultrasound imaging, identify the amniotic fluid in the phantom as the insertion target. 
	NOTE: The insertion target will depend on the context; it could include a particular region of tissue for diagnosis or therapy during a clinical procedure, or a designated location in an imaging phantom to mimic a tissue region.</li>
	<li>Insert the needle toward the insertion target. During the insertion, alternate between operation modes (imaging and tracking) continuously using the switch on the custom probe.</li>
</ol>

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The authors declare that there are no conflicts of interest.

Here we demonstrate how 3D ultrasonic tracking can be performed with a custom ultrasound imaging probe and a fiber-optic hydrophone integrated within a needle. From a clinical translation standpoint, several aspects of the custom probe developed in this study are attractive. Its compact size is well-suited for use in small spaces such as the axilla where maneuvering bulky 3D imaging probes is challenging. One limitation of the implementation of the 3D ultrasonic tracking presented here is that manual switching was required to alternate between imaging and tracking modes. In future implementations, this switching could be done directly by the ultrasound imaging system.
The fiber-optic hydrophone is well suited to ultrasonic needle tracking. Its high degree of miniaturization and flexibility allow for its integration into medical devices with small lateral dimensions. Its broad frequency bandwidth16 allows for compatibility with different clinical ultrasound probes. Additionally, its omnidirectionality16 allows for tracking needles that are inserted at a wide range of angles. Finally, its immunity to disturbances from EM fields and metal objects makes it more suitable to clinical settings in contrast to EM tracking. To achieve greater ultrasound detection sensitivity, a plano-concave Fabry-P&#233;rot cavity could be used in the future17. Ultimately, ultrasonic tracking could be combined with other modalities in a single optical fiber, such as reflectance spectroscopy18,19,20,21,22,23, Raman spectroscopy24, optical coherence tomography25,26, and photoacoustic imaging27,28,29,30,31,32,33.
Ultrasonic tracking has limitations that are shared with ultrasound imaging. First, tissue heterogeneities will negatively impact ultrasonic tracking; spatial variations in the speed of sound of tissue will decrease the tracking accuracy, as demonstrated by numerical simulations in a previous study14. Second, anatomical structures that are highly reflective to ultrasound waves, such as bony structures or air cavities, are probably not compatible with ultrasonic tracking. In future studies, the needle tip position obtained with other imaging modalities, such as 3D rotational C-arm computed X-ray tomography, could be used to assess the accuracy of 3D ultrasonic tracking in heterogeneous tissues in vivo.
Despite recent advancements in ultrasound imaging, precise tracking and efficient manipulation of medical devices under the guidance of this modality remain challenging, even for expert practitioners. Active communication between external ultrasound probes and medical devices, as shown here, could improve procedural safety and efficiency. These improvements could greatly facilitate adoption of ultrasound imaging in place of X-ray fluoroscopy in several clinical contexts, such as spinal insertions for interventional pain management. The system developed in this study enables 3D ultrasonic tracking and 2D ultrasound imaging with a compact ultrasound probe. It could improve ultrasound-guided minimally invasive procedures by providing precise localization of the needle tip within current clinical workflow.

Accurate and efficient localization of invasive medical devices is highly desired in many ultrasound-guided minimally invasive procedures. These procedures are encountered in clinical contexts such as regional anesthesia and interventional pain management1, interventional oncology2, and fetal medicine3. Visualization of the medical device tip can be challenging with ultrasound imaging. During in-plane insertions, needles often have poor visibility when insertion angles are steep. Moreover, during out-of-plane insertions, the needle shaft can be misinterpreted as the needle tip. When the needle tip is not ultrasonically visible, it can cause complications by damaging critical tissue structures.
Many methods are available to localize medical devices during ultrasound imaging, but a reliable one that is compatible with current clinical workflow is highly desired. Echogenic surfaces can be used to improve visibility during steep angle in-plane insertions4. Electromagnetic tracking systems can be used during out-of-plane insertions, but electromagnetic field disturbances can severely degrade their accuracy. 3D ultrasound imaging can improve visibility of medical devices in certain cardiac and fetal procedures when they are surrounded by fluids5. However, 3D ultrasound imaging is not widely used for needle guidance, in part due to the complexities associated with image interpretation.
Ultrasonic tracking is a method that has shown great potential for improving medical device visibility6,7,8,9,10,11,12,13,14. With ultrasonic tracking, the medical device has an embedded ultrasound sensor or transmitter that actively communicates with the external ultrasound imaging probe. The medical device position can be identified from the measured ultrasound time-of-flights between the embedded ultrasound sensor/transmitter and different transducer elements of the probe. To date, ultrasonic tracking has been limited to in-plane tracking, which has greatly restricted its clinical use.
Here, a demonstration of how 3D ultrasonic tracking can be performed with a custom ultrasound imaging probe and a fiber-optic hydrophone affixed within the cannula of a needle is provided (Figure 1). This custom probe, which was designed by the authors and manufactured externally, comprises a central array of transducer elements and four side arrays. The central array is used for 2D ultrasound imaging; the side arrays, for 3D needle tip tracking in concert with the fiber-optic ultrasound receiver. It is shown how the fiber-optic ultrasound receiver can be positioned and affixed within the needle cannula, how the tracking accuracy of the system can be measured on the benchtop, and how clinical validation can be performed.

<table><tbody><tr><td>Ultrasound imaging system</td><td>BK ultrasound (ultrasonix)</td><td>SonixMDP</td><td></td></tr><tr><td>Custom ultrasound probe</td><td>Vermon</td><td></td><td></td></tr><tr><td>Spinal needle&amp;nbsp;</td><td>Terumo</td><td></td><td>20 gauge</td></tr><tr><td>Fibre-optic hydrophone</td><td>Precision Acoustics</td><td></td><td></td></tr><tr><td>Fibre-optic stripping tool&amp;nbsp;</td><td>Thorlabs</td><td>FTS4</td><td></td></tr><tr><td>Stereo microscope&amp;nbsp;</td><td>Leica Microsystems&amp;nbsp;</td><td>Z16APO</td><td></td></tr><tr><td>Tuohy-Borst Sidearm adapter&amp;nbsp;</td><td>Cook Medical</td><td>PTBYC-RA</td><td></td></tr><tr><td>Pipette&amp;nbsp;&amp;nbsp;</td><td>Eppendorf</td><td></td><td>100 mL</td></tr><tr><td>Micropipette tip&amp;nbsp;</td><td>Eppendorf</td><td></td><td>20 &amp;micro;L</td></tr><tr><td>Ultraviolet optical adhesive&amp;nbsp;</td><td>Norland Products</td><td>NOA81</td><td></td></tr><tr><td>Syringe</td><td>Terumo</td><td></td><td>&amp;nbsp;10 mL</td></tr><tr><td>Ultraviolet light source&amp;nbsp;</td><td>Norland Products</td><td>Opticure 4 Light Gun</td><td></td></tr><tr><td>Data acquisiton card&amp;nbsp;</td><td>National Instruments</td><td>USB-5132</td><td></td></tr><tr><td>Articulated arm&amp;nbsp;</td><td>CIVCO</td><td>811-002</td><td></td></tr><tr><td>Thiopental sodium&amp;nbsp;</td><td>Novartis Animal Health UK&amp;nbsp;</td><td></td><td>Thiovet</td></tr><tr><td>Isoflurane</td><td>Merial Animal Health</td><td></td><td>Isoflurane-Vet</td></tr><tr><td>Ocular lubricant</td><td>Allergan, Marlow, UK</td><td></td><td>Lacri-Lube</td></tr><tr><td>Skin lubricant</td><td>Adams Healthcare, Garforth, UK</td><td></td><td>Hibitane 2%</td></tr></tbody></table>

three-dimensional ultrasonic needle tip tracking with a fiber-optic ultrasound receiver

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. When visualization is lost, the medical device can cause trauma to critical tissue structures. Here, a method to track the needle tip during ultrasound image-guided procedures is presented. This method involves the use of a fiber-optic ultrasound receiver that is affixed within the cannula of a medical needle to communicate ultrasonically with the external ultrasound probe. This custom probe comprises a central transducer element array and side element arrays. In addition to conventional two-dimensional (2D) B-mode ultrasound imaging provided by the central array, three-dimensional (3D) needle tip tracking is provided by the side arrays. For B-mode ultrasound imaging, a standard transmit-receive sequence with electronic beamforming is performed. For ultrasonic tracking, Golay-coded ultrasound transmissions from the 4 side arrays are received by the hydrophone sensor, and subsequently the received signals are decoded to identify the needle tip&#39;s spatial location with respect to the ultrasound imaging probe. As a preliminary validation of this method, insertions of the needle/hydrophone pair were performed in clinically realistic contexts. This novel ultrasound imaging/tracking method is compatible with current clinical workflow, and it provides reliable device tracking during in-plane and out-of-plane needle insertions.

The animal experiment was conducted in accordance with UK Home Office regulations and the Guidance for the Operation of Animals (Scientific Procedures) Act (1986). The sheep was housed in accordance with UK Home Office guidelines relating to animal welfare; the experiments were conducted under the Home Office Project License 70/7408 entitled &#34;Prenatal therapy with stem cells and gene transfer&#34;. Ethics approval for sheep experiments was provided by the University College London, United Kingdom and the Animal Welfare Ethics Review Boards of the Royal Veterinary College.
With ethics approval in place, a pregnant sheep for preclinical in vivo validation was used. After receiving intravaginal progesterone suppositories for 2 weeks, ewes were time-mated to induce ovulation, as described by David et al.34 At 130 days of gestation, one pregnant ewe was starved overnight with a pregnant companion ewe. The ewe then underwent general anesthesia induced with thiopental sodium 20 mg kg-1 intravenously and was maintained with 2-2.5% isoflurane in oxygen after intubation via a ventilator. Correct intubation was confirmed by listening to the lungs bilaterally. Anesthesia was confirmed by assessment of the corneal reflex. Oxygen saturation was measured continuously using a saturation monitor on the tongue or ear. The ewe was placed on her back in semi-recundancy and a nasogastric tube was passed to ease passage of stomach contents. An ocular lubricant was applied to the eyes to keep them moist. After clipping of the fleece, the abdomen of the ewe was double scrubbed with a skin disinfectant. Sterile coupling gel was applied to the abdomen and ultrasound examination was used to confirm the gestational age of the ewe34 and to assess fetal lie. At the end of the surgery the animal was humanely killed using an overdose of thiopental sodium (40 mg kg-1 intravenously).
The practitioner (A.L.D.) identified the umbilical cord as a target. A needle was inserted into the uterine cavity, and the tip was tracked along a trajectory that attained an out-of-plane distance of 15 mm and a depth of 38 mm (Figure 3). Golay coding improved the SNR, with a 7.5-fold increase relative to conventional bipolar excitation (Figure 3B). The 3D tracked needle tip positions were overlaid on the 2D ultrasound image using crosses with widths indicative of the out-of-plane distance and colors indicative of the imaging (step 2.6) (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57207/57207fig1.jpg" /> 
Figure 1: System overview. An ultrasound (US) imaging/tracking probe allows for both 2D US imaging and 3D needle tracking. It is driven by an US scanner that provides control over the tracking element transmissions. A switch allows for the electronic selection of transducer elements to alternate between two operation modes: imaging with the central array and tracking with side arrays. A fiber optic hydrophone (FOH) ultrasound receiver, positioned within the lumen of a 20G needle, receives transmissions from the side arrays. T/R: transmit/receive; LT: line trigger; FT: frame trigger; PC: personal computer; DAQ: data acquisition card. This figure and caption are reproduced with permission from Xia, W. et al.14. <a href="https://www.jove.com/files/ftp_upload/57207/57207fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57207/57207fig2.jpg" /> 
Figure 2: Transducer element layout of the custom ultrasound imaging probe. A central array with 128 elements and an acoustic lens enables US imaging. Side arrays, with 32 elements per row and 128 elements in total, enable 3D needle tracking. This figure and caption are reproduced with permission from Xia, W. et al.14. <a href="https://www.jove.com/files/ftp_upload/57207/57207fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57207/57207fig3.jpg" /> 
Figure 3: Needle insertion with 3D tracking in vivo. (A) Tracked needle tip positions (circles: P1-P6) obtained during an insertion into the uterine cavity of a pregnant sheep. (B) Signal-to-noise ratios (SNRs) of the tracking signals (imaging plane: X = 0). (C) Overlay of 3 of the tracked positions onto a 2D US image that was acquired with the central array. The end-to-end length of each cross corresponded to the out-of-plane distance; the color (red/yellow) corresponded to the side of the imaging plane. Key anatomical features are depicted with outlines (right). S: skin; PF: percutaneous fat; UW: uterus wall; AF: amniotic fluid; UC: umbilical cord; FA: fetal abdomen. This figure and caption are reproduced with permission from Xia, W. et al.14. <a href="https://www.jove.com/files/ftp_upload/57207/57207fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver at JoVE.com

Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver

Accurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented.

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. ...

three-dimensional-ultrasonic-needle-tip-tracking-with-fiber-optic

Research

JoVE Journal

Engineering

10.3K Views.  University College London. Nauwkeurig en efficiënt visualisatie van invasieve medische hulpmiddelen is uiterst belangrijk in vele echografie-geleide minimaal invasieve procedures. Hier wordt een methode voor het lokaliseren van de ruimtelijke positie van een naald tip ten opzichte van de echografie imaging sonde gepresenteerd.

Video: Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver

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 ...

Bioengineering

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

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 ...

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

Evaluating Targeting Accuracy in the Focal Plane for an Ultrasound-guided High-intensity Focused Ultrasound Phased-array System

Phased arrays are increasingly used as high-intensity focused ultrasound (HIFU) transducers in the existing extracorporeal ultrasound-guided HIFU (USgHIFU) systems. The HIFU transducers in such systems are usually spherical in shape with a central hole where a US imaging probe is mounted and can be rotated. The image on the plane of treatment can be reconstructed through the image sequence acquired during the rotation of the probe. Therefore, the treatment plan can be made on the reconstructed images. In order to evaluate the targeting accuracy in the focal plane of such systems, the protocol of a method using a bovine muscle and marker-embedded phantom is described. In the phantom, four solid balls at the corners of a square resin model serve as the reference markers in the reconstructed image. The target should be moved so that both its center and the center of the square model can coincide according to their relative positions in the reconstructed image. Swine muscle with a thickness of about 30 mm is placed above the phantom to mimic the beam path in clinical settings. After sonication, the treatment plane in the phantom is scanned and the boundary of the associated lesion is extracted from the scanned image. The targeting accuracy can be evaluated by measuring the distance between the centers of target and lesion, as well as three derivative parameters. This method cannot only evaluate the targeting accuracy of the target consisting of multiple focal spots rather than a single focal spot in a clinically relevant beam path of the USgHIFU phased-array system, but it can be also used in the preclinical evaluation or regular maintenance of USgHIFU systems configured with phased-array or self-focused HIFU transducer.

This study describes a protocol to evaluate the targeting accuracy in the focal plane of an ultrasound-guided high-intensity focused ultrasound phased-array system.

Phased arrays are increasingly used as high-intensity focused ultrasound (HIFU) transducers in the existing extracorporeal ultrasound-guided HIFU ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Three-Dimensional Finger Motion Tracking during Needling: A Solution for the Kinematic Analysis of Acupuncture Manipulation

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D motion tracking technology to measure the kinematic parameters of the joints of fingers during acupuncture manipulation (AM) and establish three technical indicators "amplitude, velocity and time". This method can reflect the operation characteristics of AM and provide quantitative parameters along three axes of multiple finger joints. The current evidence shows that the method has great potential for future applications such as the study of the dose-effect relationship of acupuncture, teaching, and learning of AM, and the measurement and preservation of famous acupuncturists' AM.

This experimental method describes a solution for the kinematic analysis of acupuncture manipulation with three-dimensional finger motion tracking technology.

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D ...

Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver

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