Name: How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow (Video) | JoVE Journal
Uploaded: 2010-11-11T15:10:00.000+00:00
Duration: 5 min 24 s
Description: Learn to build a Laser Speckle Contrast Imaging system for real-time blood flow monitoring in small animals. This method enables full-field visualization of vascular dynamics using a camera and diffuse laser source.

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5 minutes

The authors acknowledge support from the American Heart Association (0735136N), Dana Foundation, National Science Foundation (CBET/0737731), and the Coulter Foundation.

1. Imaging Setup
<ol>
<li>A camera with a macro zoom lens should be mounted to a vertical stage or surgical microscope (Instead of a macro zoom lens, a microscope objective and lens or a simple two lens system could be used depending on the desired magnification).</li>
<li>Download appropriate software from our website to control the camera (<a href="http://bach.bme.utexas.edu/mediawiki/index.php/Software">http://bach.bme.utexas.edu/mediawiki/index.php/Software</a>).</li>
<li>The camera software should be used to confirm an object is in focus at the desired height</li>
<li>A laser diode with a collimation kit should then be set up so that divergent laser light illuminates the object.</li>
<li>Turn down/off all ambient light to confirm that the laser light is evenly illuminating the entire field of view of the camera.</li>
<li>In this example, red laser light was used because it was easier to demonstrate how to build the system, but infrared laser light could just as easily be used and would have the additionally benefit of penetrating deeper in tissue. Also, with appropriate filters in front of the camera to block visible light, infrared light could be used with the room lights on.</li>
</ol>
2. Surgical Preparation
<ol>
<li>This is a non-survival surgery, although the experiment could be done chronically in a survival study by using a chamber window.</li>
<li>Anesthetize the animal and place it into a stereotactic frame.</li>
<li>Remove the skin and tissue surrounding the skull.</li>
<li>Using a dental drill, thin the skull over the desired brain region to transparency being careful to flush the surface with saline frequently in order to avoid damaging the brain.</li>
<li>Use dental cement to create a well around the desired region and then place a drop of mineral oil or silicone gel into the well to improve visibility.</li>
<li>Alternatively, the skull could be removed and a chamber window could be created here.</li>
</ol>
3. Collecting Data
<ol>
<li>Use the camera software to acquire images and also calculate speckle contrast values.</li>
<li>Place the animal into the field of view of the camera and adjust the camera height or lens focus until clear images of the vasculature are seen.</li>
<li>Make sure that enough laser light is reaching the camera without saturating it. Using the histogram of the image, adjust the laser power to ensure the majority of camera pixels are excited to about half their capacity.</li>
<li>Select the number of images you wish to acquire and how much averaging to do before beginning the experiment.</li>
<li>Once the experiment begins, blood flow changes can easily be monitored by selecting regions of interest or by generating an image of relative blood flow.</li>
</ol>
4. Representative Results
Figure 1 shows an example of a typical raw speckle image and a converted speckle contrast image that should be generated when using the software to examine blood flow in the brain. For visualizing changes in blood flow, it is easier to have the software generate relative maps of blood flow. Figure 2 shows a typical series of relative blood flow images during a transient increase in blood flow that travels across the field of view. The red color represents an increase in blood flow while the blue color shows a decrease. The green color indicates that there is no change in blood flow relative to a given baseline.
 <img src="/files/ftp_upload/2004/2004fig1.jpg" alt="Raw and speckle contrast images; laser speckle contrast analysis for blood flow measurement." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Example of a raw speckle image (left) and speckle contrast image (right).
 <img src="/files/ftp_upload/2004/2004fig2.jpg" alt="Relative blood flow change over time, heat map images, color scale, 15s to 120s, vascular study." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Example of several relative blood flow images at different time points during a transient increase in blood flow followed by a decrease in blood flow.

<ol><li>Briers, J. D., Fercher, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+blood-flow+visualization+by+means+of+laser+speckle+photography%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">Retinal blood-flow visualization by means of laser speckle photography.</a> Invest. Ophthalmol. Vis. Sci. 22, 255-259 (1982).</li>
<li>Boas, D. A., Dunn, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser+speckle+contrast+imaging+in+biomedical+optics%5BTitle%5D)+AND+%22J.+Biomed.+Opt%22%5BJournal%5D)">Laser speckle contrast imaging in biomedical optics.</a> J. Biomed. Opt. 15, 011109-011109 (2010).</li>
<li>Dunn, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+imaging+of+total+cerebral+hemoglobin+concentration%2C+oxygenation%2C+and+blood+flow+during+functional+activation%5BTitle%5D)+AND+%22Opt+Lett%22%5BJournal%5D)">Simultaneous imaging of total cerebral hemoglobin concentration, oxygenation, and blood flow during functional activation.</a> Opt Lett. 28, 28-30 (2003).</li>
<li>Devor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coupling+of+the+cortical+hemodynamic+response+to+cortical+and+thalamic+neuronal+activity%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Coupling of the cortical hemodynamic response to cortical and thalamic neuronal activity.</a> Proc Natl Acad Sci U S A. 102, 3822-3827 (2005).</li>
<li>Ayata, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pronounced+hypoperfusion+during+spreading+depression+in+mouse+cortex%5BTitle%5D)+AND+%22J+Cereb+Blood+Flow+Metab%22%5BJournal%5D)">Pronounced hypoperfusion during spreading depression in mouse cortex.</a> J Cereb Blood Flow Metab. 24, 1172-1182 (2004).</li>
<li>Jones, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+multispectral+reflectance+imaging+and+laser+speckle+flowmetry+of+cerebral+blood+flow+and+oxygen+metabolism+in+focal+cerebral+ischemia%5BTitle%5D)+AND+%22J.+Biomed.+Opt%22%5BJournal%5D)">Simultaneous multispectral reflectance imaging and laser speckle flowmetry of cerebral blood flow and oxygen metabolism in focal cerebral ischemia.</a> J. Biomed. Opt. 13, (2008).</li>
<li>Dunn, A. K., Bolay, H., Moskowitz, M. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+imaging+of+cerebral+blood+flow+using+laser+speckle%5BTitle%5D)+AND+%22J+Cereb+Blood+Flow+Metab%22%5BJournal%5D)">Dynamic imaging of cerebral blood flow using laser speckle.</a> J Cereb Blood Flow Metab. 21, 195-201 (2001).</li>
<li>Dunn, A. K., Devor, A., Dale, A. M., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+extent+of+oxygen+metabolism+and+hemodynamic+changes+during+functional+activation+of+the+rat+somatosensory+cortex%5BTitle%5D)+AND+%22Neuroimage%22%5BJournal%5D)">Spatial extent of oxygen metabolism and hemodynamic changes during functional activation of the rat somatosensory cortex.</a> Neuroimage. 27, 279-290 (2005).</li>
<li>Farkas, E., Bari, F., Obrenovitch, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-modal+imaging+of+anoxic+depolarization+and+hemodynamic+changes+induced+by+cardiac+arrest+in+the+rat+cerebral+cortex%5BTitle%5D)+AND+%22Neuroimage%22%5BJournal%5D)">Multi-modal imaging of anoxic depolarization and hemodynamic changes induced by cardiac arrest in the rat cerebral cortex.</a> Neuroimage. 51, 734-742 (2010).</li>
<li>Sakadzic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+imaging+of+cerebral+partial+pressure+of+oxygen+and+blood+flow+during+functional+activation+and+cortical+spreading+depression%5BTitle%5D)+AND+%22Appl.+Opt%22%5BJournal%5D)">Simultaneous imaging of cerebral partial pressure of oxygen and blood flow during functional activation and cortical spreading depression.</a> Appl. Opt. 48, (2009).</li>
<li>Ponticorvo, A., Dunn, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+imaging+of+oxygen+tension+and+blood+flow+in+animals+using+a+digital+micromirror+device%5BTitle%5D)+AND+%22Opt+Express%22%5BJournal%5D)">Simultaneous imaging of oxygen tension and blood flow in animals using a digital micromirror device.</a> Opt Express. 18, 8160-8170 (2010).</li>
</ol>

No conflicts of interest declared.

In this video we have demonstrated how easy it is to build and use a laser speckle contrast imaging (LSCI) system to look at changes in blood flow. LSCI was developed in the 1980s as a way to generate maps of blood flow in the retina1. While still used to image the retina and skin perfusion, it has become extremely popular as a technique to image blood flow in the brain2. This is largely due to the excellent spatial and temporal resolution provided and the simplicity of the instrumentation. LSCI h...

<table><tbody><tr><td>Firewire camera</td><td>Basler</td><td>scA640-74f</td><td></td></tr><tr><td>Macro zoom lens</td><td>Edmund Scientific</td><td>NT58-240</td><td></td></tr><tr><td>Laser diode</td><td>Thorlabs Inc.</td><td>HL6501MG</td><td></td></tr><tr><td>Laser diode controller</td><td>Thorlabs Inc.</td><td>LDC201CU</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;The technique is versatile enough to be used with a wide range of equipment. The only things necessary to perform the experiment are a compatible camera with a lens, a laser diode of any type with a controller, and the provided software. A table of the specific equipment used in the video is included above.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;A complete list of additional parts that can be used in this experiment is found on our website, &lt;a href=&quot;http://bach.bme.utexas.edu/mediawiki/index.php/Hardware&quot;&gt;http://bach.bme.utexas.edu/mediawiki/index.php/Hardware&lt;/a&gt;&lt;/p&gt;</td></tr></tbody></table>

how to build a laser speckle contrast imaging (lsci) system to monitor blood flow

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

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.

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

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow.  The technique analyzes ...

This video demonstrates a procedure for putting together a laser speckle contrast imaging or LSCI system capable of full field imaging of blood flow in small animals. This is accomplished by first setting up both a camera with an attached lens and a laser source that emits diffuse laser light evenly over the field of view of the camera. The second step of the procedure is to prepare a flow phantom or an animal with a suitable window for imaging the desired tissue.The final step of the procedure is to use software to collect images from your camera and turn them into speckle contrast values, ideally in real time. Ultimately, results can be obtained that show images of relative blood flow through laser speckle contrast imaging. Hi, I'm Adrian Pontecorvo from the laboratory of Dr.Andrew Dunn in the biomedical engineering department at the University of Texas at Austin.Today we'll show you a procedure for building your own laser speckle contrast imaging setup. We use this system in our lab to study cerebral blood flow. So let's get started.Laser speckle contrast imaging or LSCI is a simple and powerful technique that is used for full field imaging of blood flow. Diffuse laser light creates a speckle pattern and the speckle contrast value quantifies variations in the speckle pattern that are related to the flow of red blood cells as they pass through it. To begin building the imaging system, mount a camera with a macro zoom lens to a vertical stage, or you could mount the camera to a surgical operating microscope.Now follow the appropriate directions from our website to download the software that will be used to control the camera, place any object in our case, a simple business card into the field of view, and adjust the lens until it is in focus. You can run the software in live video mode to make sure your object is in focus. Next, set up a laser diode with a collation kit and then move it over to your object to illuminate it, turn down ambient light and adjust laser light so that it is evenly illuminating the entire field of view of the camera.Red laser light was used here to make things easier to see, but near infrared laser light could also be used and has the additional benefit of penetrating deeper in tissue. If a near infrared laser is used, make sure that your camera does not have a built-in infrared filter that would block the laser light with the imaging equipment set up. Let's see how to surgically prepare animals for experiments.Before the imaging system can be used, animals must go through a surgical preparation to create a window over the tissue of interest. This involves thinning the skull over the desired brain region. Begin by placing the urethane anesthetized animal into a stereotactic holder.Use a dissection microscope to remove the skin and tissue surrounding the skull. Now use a dental drill to thin the skull over the desired region until it is transparent. Depending on the experiment, the thin skull can either be removed or simply left alone.Next, drill a small hole about two millimeters away from the window. This is done because the experiment later involves using a needle to prick the surface of the brain. Finally, use dental cement to create a well around the desired region and place a drop of oil or silicone gel into the well to improve visibility with the animal prepared.Let's see how to collect data when collecting data. The camera software is used to acquire images and also calculate spec contrast values. Place the animal into the field of view of the camera, and with the software in live video mode, adjust either the camera height or lens focus until the desired region is seen.Change the position of the laser diode so that it is illuminating the thin skull region and adjust the laser power to make sure enough laser light is reaching the camera without saturating it. You should now be able to see clear images of the vasculature. Before you are ready to begin your experiment, select the number of images to acquire and how much averaging to do.Also, select regions of interest. You may want to monitor more closely as data is acquired. Relative blood flow changes can easily be seen by generating an image of changes to the speckle contrast value or by monitoring the speckle contrast values in the regions of interest.In this experiment, pricking the surface of the brain with the needle induces a large increase in blood flow typically associated with cortical spreading depression. We've just shown you how to build your own laser speckle contrast imaging system and use it to image cerebral blood flow in vivo in real time. When doing this procedure, it's important to remember to test your system on something simple like a business card to make sure your image is in focus and that your laser light is evenly illuminating your field of view.So that's it. Thanks for watching and good luck with your experiments.

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Research

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Neuroscience

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Diffuse Correlation Spectroscopy to Measure Cerebral Blood Flow in a Human Subject

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Learn to build a Laser Speckle Contrast Imaging system for real-time blood flow monitoring in small animals. This method enables full-field visualization of vascular dynamics using a camera and diffuse laser source.

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

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