The overall goal of this experiment is to introduce an approach to overcome movement artifact when using a laser speckle contrast imaging system for alternating speeds of blood microcirculation. This method can help researchers answer key questions in the blood dynamic responses field, in particular, when seeking to certain the effect of alternating the speeds of blood microcirculation in a single experiment. The main advantage of this technique is that it overcomes a major shortcoming of LSCI, which is high sensitivity to artifact movement.

The implications of this technique have allowed us to evaluate different modes of stimulating blood circulation. This is clinically relevant, as there is a proposed relationship between increased microcirculation and the reduction of edema in post-surgical populations, such as total hip replacement patients. This method can provide insight into the dynamics of microcirculation, and so it could also be applied to other experiments, such as those looking at monitoring devices that promote wound healing.

Generally, individuals unfamiliar with this method will struggle because LSCI is very sensitive to artifact movement, and so detection of microcirculatory changes will not be possible unless the body part is held completely still. We first had the need to develop this method when we looked at the effect of calf neuromuscular electrical stimulation and intermittent pneumatic compression on thigh microcirculation. We found that there was movement of the leg.

Visual demonstration of this method is critical, as the experimental protocol could be hard to visualize, and so therefore, difficult to replicate. To begin, connect the power supply, USB, and the FireWire interface to the rear panel of the moorFLPI LSCI. Next, assemble the desktop support arm using four screws with the moorFLPI LSCI turned upside-down and fixed to the support arm.

Once attached, rotate the mounting bracket for downward imaging. Have the participant sit in a temperature-controlled room for 10 minutes prior to any testing so that they may adapt to the room temperature. Make sure that there are no strong sources of artificial light or sunlight shining on the participant, as the ambient light could affect the moorFLPI near the infrared laser source, which operates at 780 nanometers.

Test the acceptability of the room light by opening the imaging setup window and obstructing the laser. If the image is almost completely black, then no further steps are required. Otherwise, further reduce the ambient light.

Ensure the participant is relaxed throughout the assessment. Have them place their feet flat on the ground and avoid any conversations during the test. Now, place eight square centimeters of an adhesive opaque patch on the skin area to mask the blood flow, and to account for the drawback of artifact movement.

Open the imaging software and select spatial processing for 25 frames per second of capture at 152 by 113 pixels. Then select live image measurement. Adjust the position of the imager so that it is 20 centimeters away from the participant.

Also, adjust the zoom, focus, and polarizer for minimal specular reflection. The image should appear quite flat and featureless. Set an exposure time of 20 milliseconds for high sensitivity to small changes and low flux.

Use a display rate of 25 hertz and a time constant of 0.3 seconds to account for rapid blood flow changes and to achieve optimum contrast through reducing the image noise. Next, create two equal size regions of interest named ROI 1 and ROI 2. Align ROI 2 so that it is within the eight square cenitmeters of the opaque patch on the subject's skin.

Take care so that ROIs are not interchanging, but keep them close, within two to four centimeters to reduce the need for recentering in case any mechanical movement results in ROI 2 no longer being in the opaque area. Finally, set the gain value to between 70 and 80. This will reduce the accuracy lost in low and high intensity areas.

Record the blood flow measurement in video format and save them for offline analysis using an image review module. Once recorded, open the moorFLPI image review software and load the video for analysis. Calculate the mean flux within the 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 opaque patch. When not configured correctly, the flux and raw speckle images result in high gain with poor visibility, which will result in a less accurate blood flow measurement. If configured properly, as shown in this protocol, the resulting images have maximum visibility for a reliable result.

Properly configured images result in clear definition between slow blood flow and fast blood flow. This was achieved in a continuous data recording of an alternating flux. Once mastered, this technique can be done in 20 minutes if it is performed properly.

While attempting this procedure, it's important to remember that flux measurements are done relative to a reference baseline. In the case of this methodology, the rest stage is introduced as a reference baseline. Therefore, a fast and a slow stage blood flow is expressed as a percentage change from a baseline or rest stage.

After its development, this technique paved the way for researchers in the field of blood microcirculatory analysis to explore the effectiveness of different type of devices that aim to increase microcirculatory flow. After watching this video, you should have a good understanding of how to measure alternating speeds of blood microcirculation using LSCI, and how to overcome any movement artifact that may affect the result if not properly accounted for.