The overall goal of this procedure is to measure the velocity profile of a blood solution in a microchannel using microparticle Image LOC symmetry. This is accomplished by first calibrating the system using a Newtonian solution in the micro channel to set the scale, adjust image, timing, and to find the center plane of the channel. The second step is to introduce the blood solution into the channel setup and to ensure that there are no micro bubbles.
Next, the blood flow is imaged at the desired planes in the channel, using pairs of images from a pulsed camera or video from a high speed camera. The final step is to cross correlate between the image pairs or the video sequence to obtain the displacement vector field within the channel. Ultimately, the displacement vector field can be averaged across the channel and over the timeframe of the measurement to obtain an indicative velocity profile.
This profile can be further analyzed or used for calculations. This method can answer key questions in the biomedical and biofluids field, such as the shape of the velocity profile and the sheer rate at the wall of a micro vessels. Generally, users new to this method struggle with the difficulties involved in using biological samples such as blood and the complexities involved in taking accurate measurements.
The first step is to acquire a microchannel in Polymethyl SUBOXANE or PDMS. The microchannels in this experiment are approximately 50 microns deep and 150 microns wide. Expose the fluid contacting side of the PDMS and the glass slide on which it will be bonded to oxygenated plasma for 45 seconds.
Then press them together firmly to achieve a permanent bond. In this experiment, freshly collected pore sign blood is used. To prepare it, add 0.5 grams of EDTA to two milliliters of water.
Then add the solution to one liter of warm whole blood agitated gently 10 times, and allow it to cool to room temperature when the blood is at room temperature. Centrifuge 50 milliliter blood samples at 3000 RPM for 10 minutes. Once this has been done, use a pipette to remove the plasma.
Then slowly introduce a pipette to remove the buffy coat so as to not mix it back into the blood after the buffy coat has been removed. At about 20 milliliters of phosphate buffered saline or PBS and mix it gently repeat the centrifugation removal of plasma and buffy coat and addition of PBS twice more. After the final centrifugation, remove the PBS along with the remaining buffy coat.
Take the cleaned red blood cells and suspend them in PBS or plasma at the desired concentrations. The sample here has 10%red blood cells or a hematocrit of 10. Then add one micron fluorescent tracer particles to the blood samples at the desired concentration, about 30 microliters per one milliliter of blood in this system.
Also, at this point, make a calibration solution with deionized water and the fluorescent particles. The data for this experiment are collected using a lavis laser imaging system with a pulsed camera. This is coupled with an ND YAG laser.
Before collecting data, take all necessary safety precautions for laser work. Prepare the microchip by attaching it to the syringe system using the smallest syringe possible here, 50 microliters and the shortest amount of tubing. Seal the tubing to the syringe in the chip with glue PDMS or Vaseline to reduce fluid leakage and air intake.
Be careful to avoid potential contamination of the fluid To calibrate the system first, use the calibration solution to fill the micro syringe. First, fill a standard syringe with the solution of water and fluorescing particles. Remove the plunger from the micro syringe.
Use the standard syringe to inject the calibration solution into the output of the microchannel. This will cause fluid to fill the micro syringe and escape its open-end. Continue injecting the fluid until no microbubbles are in the microchip tubing or micro syringe.
To verify the absence of microbubbles, replace the micro syringe plunger and examine the system with a microscope. Examples of microbubbles are shown here when no microbubbles are in the system, unplug the standard syringe and replace it with a syringe to collect outflow. Next, attach the micro syringe with calibration fluid to the syringe pump and level the pump.
So the tubing is horizontal. Program the syringe pump to the desired flow rate. Place the microchip on the inverted microscope stage.
Now calibrate the system. First, measure the velocity profile in the center of the channel. Also calibrate the time between pulsed images.
The particles should move between five and 10 pixels between frames. For a good correlation, be careful to measure in the middle of the channel. Once calibration is done, remove the micro syringe, plunger and outflow syringe and rinse the system with plain water.
Then fill the system with the prepared blood by using a standard syringe to inject it into the output of the micro channel. Continue until there are no micro bubbles as confirmed by microscope. Then replace the micro syringe plunger.
Next, place the micro syringe with blood in the syringe pump and level it. Keep the pump setting the same. Place the microchip on the microscope stage and start the pump.
Begin collecting data to guard against red blood cells settling in the syringe, refill it after every one or two runs. Once all the data has been collected, the images are as shown here. This is raw data obtained for 10%red blood cell concentration and a flow rate of 10 microliters per hour.
The top image is the first. The bottom image was taken 100th of a second.Later. These images are pre-processed to remove background noise after pre-processing.
Cross correlation between the images is done to obtain velocity fields. This is an example of a result in velocity profile from a system with 10%red blood cells and a flow rate of 10 microliters per hour. Note, the vertical axis is the velocity.
By using different cameras and data acquisition systems, high speed images can be taken. This can be useful for visualizing the cell-free layer in the channel and for quantifying aggregation. These images are of samples with 20%red blood cells in the top image.
The flow rate is one microliter per hour, and the blood cells are suspended in PBS, which removes their ability to aggregate. In the bottom image. The flow rate is 0.5 microliters per hour, and the blood cells are suspended in native plasma.
In this case, aggregation is visible. While attempting this procedure, it's important to remember that in focus, clear images will result in the best correlation between pairs of subsequent images. After watching this video, you should have a better understanding of how to apply microparticle image velocity symmetry to blood micro flows, despite the inherent difficulties of blood as a working fluid.
