We would like to thank Shreya Shah for her proofreading and advice.

1. Doppler phantom preparation setup (Figure 2A)
<ol>
	<li>Connect the peristaltic pump, the blood mimicking fluid reservoir, the pulse dampener and the Doppler flow phantom with the plastic tubes.</li>
	<li>Choose the canal with a 4 mm diameter.</li>
	<li>Program the pump to eject 720 mL/min of fluid for 0.3 s and then to eject 50 mL/min for 0.7 s to respectively mimic the systole and diastole cardiac phases</li>
	<li>Run the pump and gently shake the pipes to expel potential air bubbles. 
	NOTE: The operator can choose a different canal diameter and different pump rate but will have to ensure that the ultrasound sequence is fast enough to acquire the fastest flow velocities. Eq. 3 presented later can help to design the sequence.</li>
</ol>
2. Ultrafast ultrasound scanner setup (Figure 2A)
<ol>
	<li>Connect the ultrafast-enabled research scanner to the host computer with the PCI express link.</li>
	<li>Change the transducer adapter on the ultrasound scanner to match the probe connector, then connect the probe.</li>
	<li>Run Matlab and activate the ultrasound scanner license. 
	NOTE: This section and the following implicitly assume the use of a Verasonics Vantage system.</li>
</ol>
3. Ultrasound sequence programming
<ol>
	<li>Using the examples scripts, design a conventional focused &#8220;B-Mode&#8221; (i.e., echography) sequence that will be used for probe positioning.
	<ol>
		<li>Set the imaging depth to 50 mm.</li>
		<li>Set the focal depth to 35 mm.</li>
	</ol>
	</li>
	<li>Using the examples scripts, design an ultrafast ultrasound sequence.
	<ol>
		<li>Set the imaging depth to 50 mm.</li>
		<li>Program 3 tilted plane-waves at [-3,0,3] degree.</li>
		<li>Set the pulse repetition frequency (PRF) to 12 kHz.</li>
		<li>Use 4 half-cycles for the ultrasound waveform, with a center frequency depending on the probe used. A center frequency of 5.2 MHz is assumed here.</li>
		<li>Set the total duration to 1 s.</li>
	</ol>
	</li>
</ol>
4. Probe positioning and data acquisition
<ol>
	<li>Apply ultrasound gel on the probe&#8217;s lens.</li>
	<li>Place the probe on the phantom and launch the B-Mode ultrasound sequence.</li>
	<li>Locate the canal of interest. The fluid appears darker than the surrounding tissue. Place the probe in longitudinal view.</li>
	<li>Manually maintain the probe in the position of interest.</li>
	<li>End the B-Mode sequence and launch the ultrafast sequence acquisition script.</li>
</ol>
5. Image reconstruction (Figure 2B)
<ol>
	<li>Once the sequence is over, save the raw data (also called Radio-Frequency data, &#8220;RF&#8221;).</li>
	<li>Launch the image reconstruction script using the ultrasound system default software. At the end of the process, the IQ data matrix should be created. 
	NOTE: The ultrasound echoes are recorded on each element of the probe and for each emission/reception, then stored in the RF data matrix. The image reconstruction applied the appropriate delay law to each channel and results in the so-called &#8220;IQ&#8221; (In-Phase/Quadrature) matrix. The complex IQ matrix has three dimensions: two for space (image depth and width) and one for time</li>
</ol>
6. Clutter filtering (Figure 2C)
NOTE: For steps 6-7, see the Matlab script provided in the Supplementary Material.
<ol>
	<li>Reshape the 3D (space x space x time) IQ matrix into a 2D (space x time) Casorati matrix, named IQr.</li>
	<li>Compute the singular value decomposition15 of IQr (Eq. 1). 
	<img alt="Equation 1" src="/files/ftp_upload/61838/61838equ01v3.jpg" /> Eq. 1</li>
	<li>Compute the Spatial Similarity Matrix C using the spatial singular vectors U as described by Baranger et al.18 (II, D), and identify the blood subspace boundaries N.</li>
	<li>Use this cutoff N to filter the IQ data as described in Demene et al.15 (II,C).</li>
</ol>
7. Flow visualization and velocity measurements (Figure 2C)
<ol>
	<li>Compute power Doppler map PD by integrating the envelope of the filtered data IQt along the temporal dimension (Eq. 2). The 3D coordinates z, x and t are respectively the depth, width and temporal dimension, nt and is the number of frames acquired. 
	<img alt="Equation 2" src="/files/ftp_upload/61838/61838equ02v5.jpg" /> Eq. 2</li>
	<li>Display the PD map in logarithm scale. To set the dynamic range, compute the mean PD in a region outside the canal and use this value in dB as the lower bound of the dynamic range. A typical dynamic range is [-30, 0] dB.</li>
	<li>Define a circular region of interest (ROI) on the image, containing 1 to 30 pixels.</li>
	<li>Average the IQf signal over the pixels of that ROI, to obtain a vector <img alt="Equation 3" src="/files/ftp_upload/61838/61838equ03v2.jpg" /> of nt time points.</li>
	<li>Compute and display the Doppler spectrogram of <img alt="Equation 4" src="/files/ftp_upload/61838/61838equ04v2.jpg" />, using the square magnitude of the Short-Time Fourier Transform (STFT).
	<ol>
		<li>Set the STFT window to a 60-samples Hann window.</li>
		<li>Set the STFT overlap to 90% of the window length.</li>
	</ol>
	</li>
	<li>Overlay the center frequency at each time point of the spectrogram.</li>
	<li>Convert the frequency f values into blood axial velocities vz using the Doppler formula (Eq. 3). c0 is the speed of sound in the medium and fTW the center frequency of the transmitted ultrasound waveform (here 5.2 MHz). 
	<img alt="Equation 5" src="/files/ftp_upload/61838/61838equ05v4.jpg" /> Eq. 3</li>
</ol>

<ol>
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No conflict of interest

Several variations are possible around the main frame of this protocol.
Hardware concerns 
If the user supplies its custom host computer, the motherboard and the computer&#8217;s case must have an available PCI express slot. The CPU must also have enough PCIe lanes to handle all the devices.
Probe selection 
The ultrasound probe (also named transducer) is chosen according to the spatial resolution needed and to the geometry of the field of view. The higher the center frequency of the probe, the better the spatial resolution but the shorter the imaging depth. Linear, curved or phased-array probes provide field of view of respectively rectangular, circular sector and flat-top sector shapes.
Angle dependency 
Ultrafast Doppler shares the same constraint as conventional Doppler regarding the dependency to the blood flow angle. Indeed, the underlying Doppler effect only allows the detection of movement in the axial direction, meaning toward the probe surface, or away from the probe. Hence, only the axial components of the blood scatterers velocity vectors are actually measured. The true velocity can be recovered by manually indicating the local angle of the blood flow with the vertical axis, but this angle cannot always be correctly assessed. In an extreme case where the flow is perfectly orthogonal to the vertical depth axis, the Doppler effect cannot be used to reliably measure the blood velocity. More advanced techniques can measure multiple projections of the velocity vectors on several axes and can ultimately reconstruct the true velocity vector. These angle independent approaches are referred to as vector flow imaging8,9,10,24.
Aliasing 
The protocol described in this manuscript has several limitations. Firstly, the Shannon-Nyquist sampling theorem states that the maximum measurable frequency in the sampled signal cannot exceed half of the framerate. With 3 angles and a PRF of 12 kHz, the framerate is 4 kHz. Consequently, we can derive from Eq. 3 that the maximum detectable axial velocity is 30 cm/s. Considering the angle of the canal in the phantom, this framerate allows the detection of flow with velocities up to 96 cm/s. Velocities above this threshold will appear aliased in the Doppler spectrogram. For the presented setup, the peak velocities were ranging between 95 cm and 8 cm/s.
Clutter filter optimization 
Blood flow visualization strongly relies on the ability to separate the blood signals from the slowly moving tissue background. Depending on the respiration or the sonographer&#8217;s hand motion, the tissue can move with speeds similar to slow blood flow. Therefore, the so-called &#8220;clutter filter&#8221; stage aims to cancel tissue signals. The ability to detect slow blood flows only relies on the efficiency of this clutter filter stage. It has been shown that leveraging the spatiotemporal coherence of ultrafast ultrasound strongly increases the outcome of these filters. The singular value decomposition filter described by Demene et al.15 is widely used. Optimization of that method18 or more complex algorithms such as high-order SVD25, principal component pursuit26, independent component analysis27 or other low-rank decomposition28 can improve the quality of the filtered data. It is worth mentioning that in the presented in vitro setup, the only source of clutter is the hand motion of the operator. In vivo, many other factors such as respiration and arterial pulsatility are likely to induce clutter that is more predominant. In these cases, the advanced SVD filter described in this protocol becomes of prime importance.
Spectrogram interpretation 
Spectrograms are the most common tool to study blood flow characteristics in both conventional and ultrafast Doppler ultrasound. For each time point, the spectrogram shows in grayscale the velocity distribution inside the considered ROI. Complex flows such as non-laminar flows will hence naturally broaden this spectrum. However, this link between spectral broadening and velocity distribution is only true to a certain extent. It has been shown by several studies that the spectrogram width, also call spectral broadening, is indeed linked to the velocity distribution in the ROI but also to several geometrical parameters of the imaging system (array width, angles, etc)29,30,31. Therefore, while the spectrogram of a steady laminar and homogeneous flow should be a thin, flat line, it shows in practice a certain width that does not reflect the velocity distribution, but rather the geometry of the imaging setup. This potential pitfall can lead to incorrect velocity measurements. It is recommended to consider the mean velocity inside the ROI (Figure 1C dashed green line) to avoid these effects32.
3D acquisitions 
The present protocol was realized with a standard linear array transducer, resulting in 2D images. Nevertheless, 3D acquisitions can be performed, either by mechanically scanning the medium with a motorized linear probe16, or by using row-column arrays33 or matrix arrays34. The drawbacks to these new methods are the high computing costs and, for matrix probes, the need of specific scanners. An example of 3D acquisition is shown in Figure 3E.
Safety issues 
Most of the research ultrafast ultrasound scanner are not approved for clinical use. It is the responsibility of the experimenter to comply with the standards prevailing in their country, both in term of electrical safety and acoustic output. For the latter, the FDA standards35 and the IEC 62127-1 International Standard36 must be considered.
Conclusion 
In this paper, we have proposed a standard protocol to image blood flow with Ultrafast Doppler. By practicing on a calibrated flow phantom, the operator can eventually check the accuracy of their measurements. The protocol allows the user to design and execute an ultrafast ultrasound acquisition using plane-wave compounding. Ultimately, a post-processing framework is described and provides the first tool to display the blood flow profile in any region of interest of the image.

Ultrasound imaging is one of the most commonly used imaging techniques in clinical practice and research activities. The combination of ultrasound wave emission in the biological tissues followed by the recording of the backscattered echoes allows the reconstruction of anatomical images, the so-called &#8220;B-Mode&#8221;. This method is perfectly adapted for soft tissue imaging, such as biological tissues, which typically permit the penetration of ultrasound over several centimeters, with a propagation speed of &#8776;1540 m/s. Depending on the center frequency of the ultrasound probe, images with a resolution from 30 &#181;m to 1 mm are obtained. Furthermore, it is well known that the motion of an acoustic source, affects the physical characteristics of the associated waves. Particularly, the link between the frequency shifts of a wave relative to the speed of its source is described as the Doppler effect1, whose simplest manifestation is the changing siren&#8217;s pitch of a moving ambulance. Ultrasound imaging has long used this physical effect to observe the moving red blood cells2, and it proposes a variety of imaging modes commonly labelled &#8220;Doppler imaging&#8221;. These modes enable the assessment of blood flows in very different applications and organs, such as brain, heart, kidney or peripheral arteries.
Remarkably, most of the currently available ultrasound systems rely on the same technology, referred to as conventional ultrasound. The underlying principles are the following: an acoustic beam insonifies the field of view and is swept along the ultrasound transducer aperture. For each position of the beam, the echoes are recorded and converted into a line of the final image. By progressively moving the beam along the transducer, the whole field of view can be imaged line-per-line (Figure 1, left panel). This strategy was well adapted to the electrical constraints and computing power prevailing until the beginning of the 21st century. Nonetheless, it has several drawbacks. Among these, the final framerate is limited to a few hundred images per second by the beam scanning process. In terms of blood flow, this relatively low framerate affects the maximum flow velocities that can be detected, which is dictated by the sampling criteria of Shannon-Nyquist3. Moreover, conventional Doppler must deal with a complex tradeoff. In order to assess the blood flow velocity in a particular region of interest (ROI), several echoes coming from that ROI have to be successively recorded. This implies that the ultrasound beam is temporarily maintained in a fixed position. The longer the echo ensemble, the better the velocity estimation will be for that ROI. However, to produce a complete image of the field of view, the beam must scan the medium. Therefore, one can sense the conflict between these two constraints: holding the beam to precisely assess the velocity along one line, or moving the beam to produce an image. The different conventional Doppler modes (i.e., Color Doppler or Pulse Wave Doppler) directly reflect this tradeoff. Typically, the Color Doppler produces a low-fidelity flow map used for localizing the vessels4, and the Pulse Wave Doppler is then used to accurately quantify the flow in a previously identified vessel5.
These two limitations (low framerate and localization/quantification tradeoff) are overcome with very high-framerate emerging techniques. Among these, the synthetic aperture approach6 or the multiline transmit technique can be cited7. In this study, we focus on the so-called Ultrafast ultrasound method. Introduced two decades ago8,9,10, this method also relies on the emission/reception of ultrasounds, but with a radically different pattern. Indeed, instead of using a scanning focused beam, ultrafast imaging uses plane wave or diverging waves, which are able to insonify the field of view with a single emission. Following that single emission, the associated electronics is also able to receive and process the huge number of echoes originating from the whole field of view. At the end, an image can be reconstructed from a single emission/reception pattern11 (Figure 1, right panel). These unfocused emissions can have a low signal to noise ratio (SNR) due to the spread of the acoustical energy. This can be tackled by emitting several titled plane-waves (or diverging waves with different sources) and by adding the resulting images. This method is named &#8220;coherent compounding&#8221;12. Two major consequences arise. Firstly, the framerate only depends on the ultrasound time of flight and can reach typical values from 1 to 10 kHz. Secondly, this ensures the data continuity in both spatial and temporal dimensions, also referred to as spatiotemporal coherence. The conventional localization/quantification tradeoff is thus broken. This combination of a high framerate and spatiotemporal coherence has a tremendous impact on the ability to detect blood flows with ultrasound. As compared to conventional ultrasound, ultrafast ultrasound provides full characterization of the blood flow3. Practically, the user has access to the velocity time course in every pixel of the image, for the whole duration of the acquisition (typically &#8776;1 s), with a timescale given by the framerate (typically, a framerate of 5 kHz for a temporal resolution of 200 &#181;s). This high framerate makes the method suitable for a wide range of application such as fast flow in moving organs like heart chambers13 or myocardium with the coronary micro-perfusion14. Furthermore, it has been shown that its spatiotemporal coherence strongly improves its ability to separate slow blood flow from background moving tissues, therefore increasing the sensitivity to micro-vascular flow15. This capacity gives access to the micro vasculature of the brain in both animals16 and humans17.
Hence, ultrafast ultrasound is well suited to image blood flow in a variety of situations. It is restricted to soft biological tissues and will be strongly affected by the presence of hard interfaces such as bones, or gas cavity such as the lung. The tuning of the physical parameters of the ultrasound sequence allows the study of both slow (down to 1 mm/s11,16) and fast flows (up to several m/s). A tradeoff exists between the spatial resolution and the depth of penetration. Typically, a resolution of 50 &#181;m can be achieved at the cost of a penetration around 5 mm. Conversely, the penetration can be extended to 15-20 cm at the cost of a resolution of 1 mm. It is worth noting that most ultrafast scanners such as the one used in this article only provide 2D images.
Here, we propose a simple protocol to introduce the concept of Ultrafast Doppler imaging, using a programmable research ultrasound scanner and Doppler phantom mimicking a vessel (artery or vein) embedded in biological tissue.

<table><tbody><tr><td>Blood-mimicking fluid</td><td>CIRS Inc, Norfolk, Virginia, USA</td><td>069DTF</td><td></td></tr><tr><td>Doppler flow phantom</td><td>CIRS Inc, Norfolk, Virginia, USA</td><td>ATS523A</td><td></td></tr><tr><td>Matlab</td><td>MathWorks, Natick, Massachusetts, United States</td><td></td><td></td></tr><tr><td>Peristaltic pump / Doppler flow pump</td><td>CIRS Inc, Norfolk, Virginia, USA</td><td>769</td><td>Include tubings and pulse dampener</td></tr><tr><td>Transducer adpter</td><td>Verasonics, Kirkland, Washington, USA</td><td>UTA 408-GE</td><td></td></tr><tr><td>Ultrafast ultrasound research scanner</td><td>Verasonics, Kirkland, Washington, USA</td><td>Vantage 256</td><td></td></tr><tr><td>Ultrasound probe/transducer</td><td>GE Healthcare</td><td>GE 9L-D</td><td></td></tr></tbody></table>

blood flow imaging with ultrafast doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

The quality of the acquisition and the post-processing is firstly assessed by visual inspection. The shape of the canal must be clearly visible in the power Doppler image, and the tissue area must appear dark. If the power Doppler signal is not restricted to the canal, it can mean that either the clutter filter step went wrong (SVD threshold is too low), or the probe experienced a strong movement during the acquisition.
After visual inspection, the study of the spectrogram inside the canal can provide good information on the success or failure of the experiment. The spectrogram should be one-sided (all the values above or below zeros). If the spectrogram is two-sided, aliasing is present. In that case, either the flow is too fast, or the PRF is too low.
If these quality criteria are met, the blood velocities can be extracted from any ROI in the image (Figure 2C). Tuning the size of the ROI allows more or less averaging of the signals. The velocity time course of a given ROI can then be used for multiple analysis such as the computation of resistivity indexes19, wall shear stress estimation20, reactive hyperemia quantification14 and much more21,22.
Figure 3A-D shows the transposition of this protocol to various in vivo applications. In particular, the neonate brain acquisition (Figure 3B) exhibits vessels with very different flow characteristics, from small cortical venules and arterioles to the major pericallosal artery. Figure 3D illustrates the ability of ultrafast Doppler to extract blood flow signal in a strongly moving organ such as the myocardium.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61838/61838fig01.jpg" /> 
Figure 1: Conventional and Ultrafast Ultrasound Imaging.&#160;Legend: (Left) Conventional imaging with focused emission. (Right) Ultrafast imaging with plane wave emission. (adapted from Villemain et al.22). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61838/61838fig01large.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/61838/61838fig02.jpg" /> 
Figure 2: Ultrafast Doppler protocol workflow.&#160;(A) Experimental setup including the ultrafast-enabled scanner and the Doppler flow phantom. The dashed rectangle on the phantom indicates the footprint of the ultrasound transducer. (B) Automated data acquisition chain and post-processing triggered by a simple user button-press. (C) (Top) Extraction of blood flow signal and suppression of the tissue background noise (&#8220;clutter filter&#8221;) and display of the blood spectrogram in any ROI of the field of view. (Bottom) Spectrogram showing the blood velocity distribution in the ROI at different time points. The mean velocity in the ROI is traced in dashed green. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61838/61838fig02large.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/61838/61838fig03.jpg" /> 
Figure 3: Power Doppler images.&#160;Ultrafast Doppler acquisitions on several organs. (A) Adult transplanted kidney, (B) Sagittal view of a human neonate brain, (C) Adult thyroid, (D) Intramural coronary vasculature in open-chest swine experiments, (E) 3D directional power Doppler of the carotid artery and the jugular vein of a healthy volunteer (blue = descending flow, red = ascending flow). Several spectrograms are extracted for different ROI. (A-C are adapted from Baranger et al.18, D is adapted from Maresca et al.14, E is adapted from Provost et al.23). For each acquisition, the center frequency, number of angles, PRF and maximum depth was tuned according to the situation. The dynamic range for panel A, B and C are respectively -27, -35 and -30 dB. It was not provided for panel D and E. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61838/61838fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Materials. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61838/JOVE_SupScript.m" target="_blank">Please click here to download these files.</a>

Watch this Scientific Journal Video about Blood Flow Imaging with Ultrafast Doppler at JoVE.com

Blood Flow Imaging with Ultrafast Doppler

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

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7.5K Views.  The Hospital for Sick Children, PGCRL Research Institute. This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every ≈0.3 ms (depending on the ultrasound time of flight).

Video: Blood Flow Imaging with Ultrafast Doppler

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Medicine

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

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

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF).
With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment.&#160;

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

A Novel Approach to Overcome Movement Artifact When Using a Laser Speckle Contrast Imaging System for Alternating Speeds of Blood Microcirculation

The laser speckle contrast imager (LSCI) provides a powerful yet simple technique for measuring microcirculatory blood flow. Ideal for blood dynamic responses, the LSCI is used in the same way as a conventional Laser Doppler Imager (LDI). However, with a maximum skin depth of approximately 1 mm, the LSCI is designed to focus on mainly superficial blood flow. It is used to measure skin surface areas of up to 15 cm x 20 cm. The new technique introduced in this paper accounts for alternating speeds of microcirculations; i.e. both slow and fast flow flux measurement using the LSCI. The novel technique also overcomes LSCI&#39;s biggest shortcoming, which is high sensitivity to artifact movement. An adhesive opaque patch (AOP) is introduced for satisfactory recording of microcirculatory blood flow, by subtracting the LSCI signal from the AOP from the laser speckle skin signal. The optimal setting is also defined because the LSCI is most powerful when flux changes are measured relative to a reference baseline, with blood microcirculatory flux expressed as a percentage change from the baseline. These changes may be used for analyzing the status of the blood flow system.

This study introduces a novel technique for the measurement and analysis of alternating speeds of blood microcirculation in a single experiment using a laser speckle contrast imager.

The laser speckle contrast imager (LSCI) provides a powerful yet simple technique for measuring microcirculatory blood flow. Ideal for blood dynamic ...

Assessing Leg Blood Flow in Training Workloads

Doppler Ultrasound-Based Leg Blood Flow Assessment During Single-Leg Knee-Extensor Exercise in an Uncontrolled Setting

Doppler ultrasound has revolutionized the assessment of organ blood flow and is widely used in research and clinical settings. While Doppler ultrasound-based assessment of contracting leg muscle blood flow is common in human studies, the reliability of this method requires further investigation. Therefore, this study aimed to investigate the within-day test-retest, between-day test-retest, and inter-rater reliability of Doppler ultrasound for assessing leg blood flow during rest and graded single-leg knee-extensions (0 W, 6 W, 12 W, and 18 W), with the ultrasound probe being removed between measurements. The study included thirty healthy subjects (age: 33 &#177; 9.3, male/female: 14/16) who visited the laboratory on two different experimental days separated by 10 days. The study did not control for major confounders such as nutritional state, time of day, or hormonal status. Across different exercise intensities, the results demonstrated high within-day reliability with a coefficient of variation (CV) ranging from 4.0% to 4.3%, acceptable between-day reliability with a CV ranging from 10.1% to 20.2%, and inter-rater reliability with a CV ranging from 17.9% to 26.8%. Therefore, in a real-life clinical scenario where controlling various environmental factors is challenging, Doppler ultrasound can be used to determine leg blood flow during submaximal single-leg knee-extensor exercise with high within-day reliability and acceptable between-day reliability when performed by the same sonographer.

Author Spotlight: Assessing the Reliability of Doppler Ultrasound in Measuring Leg Blood Flow 

This test-retest study evaluated leg blood flow measured by the Doppler ultrasound technique during single-leg knee-extensor exercise. The within-day, between-day, and inter-rater reliability of the method was investigated. The approach demonstrated high within-day and acceptable between-day reliability. However, the inter-rater reliability was unacceptably low during rest and at low workloads.

Doppler ultrasound has revolutionized the assessment of organ blood flow and is widely used in research and clinical settings. While Doppler ...

Ultrafast Doppler Ultrasound for Hepatic Microvasculature Analysis in Mice

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.

Ultrafast Doppler ultrasound (UFUS) with high spatial resolution (100 &#956;m) and sensitivity represents a suitable noninvasive imaging modality for obtaining a comprehensive qualitative overview of the hepatic vasculature. UFUS also facilitates quantitative measurements of the microvasculature, aiming to enhance our understanding of vascular disease mechanisms.

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the ...

Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood supply. The capillary lamina of the choroid lines the outer surface of the retina and is the dominating source of oxygen in most vertebrate retinas. However, this vascular bed is challenging to image with traditional optical techniques due to its position behind the highly light-absorbing retina. Here we describe a high-frequency ultrasound technique with subsequent flow-enhancement to image deep vascular beds (0.5-3 cm) of the eye with a high spatiotemporal resolution. This non-invasive method works well in species with nucleated red blood cells (non-mammalian and fetal animal models). It allows for the generation of non-invasive three-dimensional angiographies without the use of contrast agents, and it is independent of blood flow angles with a higher sensitivity than Doppler-based ultrasound imaging techniques.

We present a non-invasive ultrasound technique for generating three-dimensional angiographies in the eye without the use of contrast agents.

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood ...

Biology

High-frequency Ultrasound Imaging of the Abdominal Aorta

Source: Amelia R. Adelsperger, Evan H. Phillips, and <a href="https://www-jove-com.bdigitaluss.remotexs.co/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
High-frequency ultrasound systems are used to acquire high resolution images. Here, the use of a state-of-the-art system will be demonstrated to image the morphology and hemodynamics of small pulsatile arteries and veins found in mice and rats. Ultrasound is a relatively inexpensive, portable, and versatile method for the noninvasive assessment of vessels in humans as well as large and small animals. These are several key advantages that ultraound offers compared to other techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), and near-infrared fluorescence tomography (NIRF). CT requires ionizing radiation and MRI can be prohibitively expensive and even impractical in some scenarios. NIRF, on the other hand, is limited by the penetration depth of light required to excite the fluorescent contrast agents.
Ultrasound has limitations in terms of imaging depth; however, this may be overcome by sacrificing resolution and using a lower frequency transducer. Abdominal gas and excess body weight can severely diminish image quality. In the first case, the propagation of sound waves is limited, while in the latter case, they are attenuated by overlying tissues, such as fat and connective tissue. As a result, no contrast or faint contrast may be observed. Finally, ultrasound is a highly user-dependent technique, requiring the sonographer to be familiar with anatomy and to be able to work around issues, such as the appearance of imaging artifacts or acoustic interference.

High-frequency Ultrasound Imaging and Image Acquisition

Source: Amelia R. Adelsperger, Evan H. Phillips, and Craig J. Goergen, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, 
...