The authors have no acknowledgements.

The method reported was utilised in a study that received ethical approval from the Bournemouth University Research Ethics Committee on the 9th February 2016 (Reference 10571).
1. LSCI Set up
<ol>
	<li>Connect the moorFLPI LSCI rear panel to its three sockets (power supply, Universal Serial Bus (USB) and IEEE1394) for the system to function.</li>
	<li>Assemble the Desktop Support Arm using 4 screws with the moorFLPI LSCI turned upside down, and fixed to the support arm.</li>
	<li>Rotate the mounting bracket for downward imaging when attached. 
	NOTE: The LSCI has three controls: (1) zoom adjuster &#8211; with the imager set in a position, less magnification can be adjusted to maximum and minimum settings for small and larger fields of view respectively. To ensure repeatability, an indexing ring label is provided; (2) focus adjuster &#8211; this is dependent on the measurement distance and must be adjusted after image position has been set. To ensure repeatability, an indexing ring label is provided; and (3) a polarizer &#8211; a linear polarizing filter is available to minimize specular reflection from exposed organs &#8211; the rotating mount can be turned through 360&#176;.</li>
	<li>Install the software to control the camera. The software is divided into two modules providing a measurement and review function.</li>
</ol>
2. Participant Preparation
<ol>
	<li>Ensure the assessment is performed in a temperature controlled room (22 &#177; 1 &#176;C) and that the participants are seated for 10 minutes prior to testing to adapt to room temperature.</li>
	<li>Avoid a strong source of artificial light and the sunlight shining on the participant or the LSCI as ambient light could affect the moorFLPI near infrared laser source operating at 785 nm. 
	NOTE: A simple test to confirm whether ambient light levels are acceptable is by opening the imaging setup window and obstructing the laser. If the image is almost completely black then no further steps are required; if there is still too much ambient light present, further action is required.</li>
	<li>Ensure the participants are relaxed throughout the assessment, with their feet flat on the ground, if seated, and avoid conversations.</li>
	<li>Place 8 cm2 of adhesive opaque patch (AOP) (e.g., Leukotape) on the skin area to mask the blood flow. This is done to account for the drawback of LSCI in terms of high sensitivity to artifact movement, and signal backscatter will be used for measurement of microcirculatory blood flow.</li>
</ol>
3. Microcirculation Image Measurement
<ol>
	<li>Select &#39;Spatial Processing&#39; for 25 frames per second capture at 152 x 113 pixels.</li>
	<li>Choose &#39;Live image measurement&#39; and adjust the position of the imager 20 cm away from the participant followed by adjusting the zoom, focus and polarizer for minimal specular reflection. The image should appear quite &#39;flat&#39; and featureless.</li>
	<li>Set an exposure time of 20 ms for high sensitivity to small changes and low flux.</li>
	<li>Use a display rate of 25 Hz and a time constant of 0.3 s to account for rapid blood flow changes, and to achieve optimum contrast through reducing the image noise.</li>
	<li>Create two equal size (2 cm2) regions of interest (ROI), named ROI 1 and ROI 2. Align ROI 2 so it is within the 8 cm2 AOP. Take care so that ROIs are not interchanging but kept close within 2-4 cm to reduce the need for re-centering if any mechanical movement results in ROI 2 no longer being in the AO area. 
	NOTE: Blood flow measurement will be less accurate in low and high intensity areas, so it is important to have an optimal gain setting. The gain value ranges between 0 - 200. An optimal gain setting value is achieved in range of 70 - 80.</li>
	<li>Perform flux measurements relative to a reference baseline. In the case of this methodology, introduce &#39;the rest&#39; stage as a reference baseline. Therefore, express a &#39;fast&#39; and &#39;slow&#39; stage blood flow as a percentage change from a baseline, &#39;rest&#39; stage.</li>
	<li>Record blood flow measurement in video format and save for offline analysis using an image review module.</li>
</ol>
4. Offline Analysis
NOTE: The moorFLPI Image Review Software allows the opening of a video to perform analysis.
<ol>
	<li>Calculate the mean flux within ROIs following a series of recordings of mean blood flow. ROI 1 is the real measurement of skin blood flow and ROI 2 is the backscattered laser speckle skin signal from the AOP.</li>
	<li>Calculate the mean blood flow by subtracting ROI 2 from ROI 1 (skin blood flow). 
	ROI 1 - ROI 2 = Mean Blood Flow</li>
</ol>

<ol>
	<li>Mahe, G., Rousseau, P., Durand, S., Leftheriotis, G., Abraham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+speckle+contract+imaging+accurately+measures+blood+flow+over+moving+skin+surfaces.">Laser speckle contract imaging accurately measures blood flow over moving skin surfaces.</a> Microvas Res. 81 (2), 183-188 (2010).</li><li>Rousseau, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+&amp;#34;region+of+interest&amp;#34;+and+&amp;#34;time+of+interest&amp;#34;,+both+reduce+the+variability+of+blood+flow+measurements+using+laser+speckle+contrast+imaging.">Increasing the &amp;#34;region of interest&amp;#34; and &amp;#34;time of interest&amp;#34;, both reduce the variability of blood flow measurements using laser speckle contrast imaging.</a> Microvas Res. 82, 88-91 (2011).</li><li>Hecht, N., Woitzik, J., Dreier, J., Vajkoczy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraoperative+monitoring+of+cerebral+blood+flow+by+laser+speckle+contrast+analysis.">Intraoperative monitoring of cerebral blood flow by laser speckle contrast analysis.</a> Neurosurg Focus. 27 (4), 1-6 (2009).</li><li>Mahe, G., Durand, S., Humeau-Heurtier, A., Leftheriotis, G., Abrham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+experimental+conditions+on+noncontact+laser+recordings+in+microvascular+studies.">Impact of experimental conditions on noncontact laser recordings in microvascular studies.</a> Microcirculation. 19 (8), 669-675 (2012).</li><li>Cheng, H., Duong, T. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+laser-speckle-imaging+analysis+method+and+its+application+to+retinal+blood+flow+imaging.">Simplified laser-speckle-imaging analysis method and its application to retinal blood flow imaging.</a> Opt Lett. 32 (15), 2188-2190 (2007).</li><li>Doherty, J., McNamara, P., Clancy, N. T., Enfield, J. G., Leahy, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+instruments+for+investigation+of+microcirculatory+blood+flow+and+red+blood+cell+concentration.">Comparison of instruments for investigation of microcirculatory blood flow and red blood cell concentration.</a> J Biomed Opt. 14 (3), 034025 (2009).</li><li>Briers, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+speckle+contrast+imaging+for+measuring+blood+flow.">Laser speckle contrast imaging for measuring blood flow.</a> Opt Appl. 37 (1), 139-152 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> moorFLPI User Manual. (8), (2012).</li><li>Mahe, G., Durand, S., Humeau-Heurtier, A., Abraham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Experimental+Conditions+on+Noncontact+Laser+Recordings+in+Microvascular+Studies.">Impact of Experimental Conditions on Noncontact Laser Recordings in Microvascular Studies.</a> Microcirculation. 19, 669-675 (2012).</li><li>Bahadori, S., Immins, T., Wainwright, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+calf+neuromuscular+electrical+stimulation+and+intermittent+pneumatic+compression+on+thigh+microcirculation.">The effect of calf neuromuscular electrical stimulation and intermittent pneumatic compression on thigh microcirculation.</a> Micro Res. 111, 37-41 (2017).</li><li>Omarjee, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+movement+detection+and+artifact+removal+during+laser+speckle+contrast+imaging.">Optimisation of movement detection and artifact removal during laser speckle contrast imaging.</a> Miscrovas Res. 97 (1), 75-80 (2015).</li></ol>

The authors have nothing to disclose.

The aim of this study was to introduce a novel technique for the measurement and analysis of alternating speeds of blood microcirculation in a single experiment using the LSCI. The measurements can be affected by ambient light, vibration and participant movement including breathing and twitching. The steps outlined in the protocol have all been designed to minimize those effects and obtain reliable and repeatable measurements of blood microcirculation.
It is emphasized that every step within the protocol is critical for an accurate measurement of blood microcirculation, the technique introduced was discovered following sequential testing of all possible setting options and combinations available including: time constant, zoom setting, exposure time, display rate, gain and flux image palette. Results were analyzed and repeated using live video display and offline analysis to find the optimum imaging setup. This was essential as the image processing software uses the fact that high perfusion produces rapid variation in laser speckle pattern, and as a result a low contrast area of well-defined speckle is produced in the video image. Perfusion image is then created in a color-coded map of microcirculatory perfusion.
Experimental area and participation preparation is found to be essential, and this can be controlled by avoiding work near sources of daylight (window) or strong sources of artificial light, as these may interfere with the moorFLPI near infra-red laser source. The protocol also introduced an AOP as it was acknowledged that the environmental vibration and movement of participant both generate signals that are indistinguishable from blood flow. The AOP proved to be a simple yet effective choice, offering a thin but opaque, light and accessible option which had a microscopically rough surface area to avoid significant specular reflection. Preliminary research by Omarjee et al.11 highlights a potential limitation by which Leukotape creates a reflection signal amplitude different to that of the skin and varies significantly between subjects; however Mahe et al.1 found no drastic difference between participants. Although Leukotape is more accessible than other bespoke, bilayer adhesive patches, the accuracy of Equation (1) measurement could be enhanced by using an alternative AOP.
The offline analysis section highlighted the importance of sizes of ROIs, and their location within the area of interest. Initially, a bigger ROI 1, approximately 8 cm2, was tried which overlaid the ROI 2. This methodology soon became un-reliable due to artifact movement resulting in the ROI 2 moving and the live experiment had to be stopped in order to re-center the ROI 2. Another short coming was that due to ROI 1 overlaying the AOP, the mean flux no longer took the area under AOP into an account, as there was no longer a backscattered signal. This meant a large section of blood microcirculation was being overlooked and therefore resulting flux data were not correct. Therefore, a methodology in which two ROIs of 2 cm2, with an AOP of 8 cm2 and no interaction between ROI 1 and ROI 2 (but kept within 2-4 cm of each other), provides a reliable and repeatable analysis technique for measurement of blood microcirculation.

Laser Speckle Contrast Imaging (LSCI) is a proven non-contact, real time monitoring method to analyse blood flow microcirculation1,2,3,4,5,6,7. The LSCI used in this paper is moorFLPI Full Field. The measuring of blood flow perfusion in large areas at high spatial and temporal resolution using a phenomenon called &#34;laser speckle&#34; is one of the main advantages of this device6. The real-time assessment of microcirculation is done through captured patterns via a camera using scattered speckle patterns. Given that the moorFLPI LSCI is intended for clinical and physiological research, the image processing software works on the fact that high perfusion produces rapid variation in the laser speckle pattern, which is then integrated by the Charged Coupled Device (CCD) to produce an area of low contrast8. The contrast is quantified and the resulting flux is colour coded to produce a perfusion image8.
Unfortunately, the LSCI is very sensitive to environmental vibration, artifact and movement of the subject area9. To date this has provided challenging when alternating flow states have been studied. This paper explains details of the explicit technique outlined in a recent study10 where a Neuromuscular Electrical Stimulation Device was used to measure blood microcirculation when there was movement of the limb being examined.

<table><tbody><tr><td>moorFLPI LSCI</td><td>Moor Instruments</td><td>Not Available - Online Link Provided in descreption</td><td>moorFLPI is an instrument designed for the measurment of blood flow within microvasculature by using infra red laser speckle contrast analysis.&amp;nbsp; https://gb.moor.co.uk/</td></tr><tr><td>moorFLPI Image Review Module</td><td>Moor Instruments</td><td>No Available - Online Link Provided</td><td>Used with moorFLPI, user can record and measure changes in blood flow by changerating a colour coded map of tissue perfusion.&amp;nbsp; https://gb.moor.co.uk/</td></tr><tr><td>Leukotape</td><td>BSN Medical</td><td>72978-10</td><td>Medical tape with microporous surface. http://www.bsnmedical.co.uk/fileadmin/z-countries/United_Kingdom/PDF/L/Leukotape_K_A46PP_low_res_11112013.pdf</td></tr></tbody></table>

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.

The LSCI experimental set up is outlined in Figure 1 with functional tools identified. A typical participant preparation for a measurement of blood flow on an area of the anterior thigh is illustrated. Adjustable mounting bracket allows rotation of the LSCI for the measurement of blood flow within microvasculature of any particular skin area. Figure 2 outlines an example of a typical raw speckle image and converted speckle image with bespoke settings outlined in the protocol for measurement of blood microcirculation.
Figure 3 shows an example of skin area and placement of AOP (step 3.1), LSCI raw imaging setup (step 3.2), a live image for slow blood flow (step 3.3) and a live image for a fast blood flow (step 3.4) achieved in a continuous data recording of an alternating flux using moorFLPI LSCI. Palette color-coding setting allows differentiation between flux levels. With the standard palette of 16 colors, low flux is seen as blue, medium flux values are seen as green and high flux values are seen as orange and red.&#160;
Skin blood flow is expressed in laser speckle perfusion units (LSPU). Figure 4 shows the graphical representation of ROI 1, ROI 2 and AOP on an area of skin. The mean blood flow is calculated in offline analysis using data from ROI 1 and ROI 2, and Equation (1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56415/56415fig1.jpg" /> 
Figure 1: moorFLPI LSCI experimental set up. Desktop support arm, cable outputs, position controls (zoom adjuster, focus adjuster and polarizer), AOP and laptop for configuration of image setting. <a href="https://cloudflare.jove.com/files/ftp_upload/56415/56415fig1large.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/56415/56415fig2.jpg" /> 
Figure 2: Raw speckle image prior to data recording. 2.1 - 2.2) Flux and raw speckle images of a badly configured setting resulting in a high gain with poor visibility which will result in a less accurate blood flow measurement. 2.3 - 2.4) System configured as per protocol, resulting in a correct gain with maximum visibility for a reliable result. <a href="https://cloudflare.jove.com/files/ftp_upload/56415/56415fig2large.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/56415/56415fig3.jpg" /> 
Figure 3: An overview of setup and record measurement using moorFLPI LSCI. 3.1. An area of skin (thigh) with a 2 cm2 AOP to account for artifact movement. 3.2) A raw speckle &#39;flat and featureless&#39; image indicating good backscatter light intensity with optimal setting. 3.3) A live image recording of a slow blood flow. 3.4) A live image recording of a fast blood flow. <a href="https://cloudflare.jove.com/files/ftp_upload/56415/56415fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56415/56415fig4.jpg" /> 
Figure 4: Graphical representation of the ROI 1, ROI 2 and AOP layout on an area of the skin. <a href="https://cloudflare.jove.com/files/ftp_upload/56415/56415fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Rest (Baseline Reference) (LSPU)</td>
			<td colspan="4">Slow Blood Flow (LSPU)</td>
			<td colspan="4">Moderate Blood Flow (LSPU)</td>
			<td colspan="4">Fast Blood Flow (LSPU)</td>
		</tr>
		<tr>
			<td>Mean Flux - ROI 1</td>
			<td>Mean Flux - ROI 2</td>
			<td>Mean Blood Flow</td>
			<td>Mean Flux - ROI 1</td>
			<td>Mean Flux - ROI 2</td>
			<td>Mean Blood Flow</td>
			<td>Blood Flow Increase % from Baseline</td>
			<td>Mean Flux - ROI 1</td>
			<td>Mean Flux - ROI 2</td>
			<td>Mean Blood Flow</td>
			<td>Blood Flow Increase % from Baseline</td>
			<td>Mean Flux - ROI 1</td>
			<td>Mean Flux - ROI 2</td>
			<td>Mean Blood Flow</td>
			<td>Blood Flow Increase % from Baseline</td>
		</tr>
		<tr>
			<td>157.9</td>
			<td>35.1</td>
			<td>122.8</td>
			<td>178.5</td>
			<td>41.6</td>
			<td>136.9</td>
			<td>10.9</td>
			<td>216.9</td>
			<td>44.6</td>
			<td>172.3</td>
			<td>33.5</td>
			<td>418.9</td>
			<td>77.5</td>
			<td>341.4</td>
			<td>94.2</td>
		</tr>
	</tbody>
</table>
Table 1: Mean flux in LSPU for ROI 1 and ROI 2 at baseline, slow, moderate and fast Blood Flow. Blood flow increase is expressed as a percentage change from a baseline stage.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56415/56415fig5.jpg" /> 
Figure 5: Example of ROI 1 and ROI 2 positioning on a skin surface area (thigh). A 16-color palette outlines the lavers of perfusions. Data is recorded for ROI 1 and ROI 2 in perfusion units and subtracted as explained in equation 1 for measurement of blood microcirculation. <a href="https://cloudflare.jove.com/files/ftp_upload/56415/56415fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Novel Approach to Overcome Movement Artifact When Using a Laser Speckle Contrast Imaging System for Alternating Speeds of Blood Microcirculation at JoVE.com

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

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 ...

a-novel-approach-to-overcome-movement-artifact-when-using-laser

Research

JoVE Journal

Medicine

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

How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow

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How to Build a Laser Speckle Contrast Imaging LSCI System to Monitor Blood Flow

This video demonstrates how to build a Laser Speckle Contrast Imaging (LSCI) system that can easily be used to monitor blood flow.

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Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

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This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source&#160;image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces&#160;images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids&#160;that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, ...

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.

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 ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

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

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