This method can help answer key questions in the field of bioengineering, specifically those pertaining to hemodynamics and their interactions with intervascular devices. The main advantage of this technique is that it makes use of equipment already found in most bioengineering laboratories and this can help reduce the barrier to entry for non-experts. Mix the PDMS prepolymer base in curing agent in a 10 to one ratio by weight.
A 66 gram mixture provides sufficient material for the fabrication of phantoms with volumes up to 50 cubic centimeters. Place the mixture in a vacuum desiccator for 60 minutes to de-gas and minimize the bubble entrapment. Use cyclic pressurization depressurization to facilitate bubble rupture.
To perform casting mount the printed ABS mold on a glass slide using molding putty to seal the interface. Carefully pour the PDMS mixture into the mold while trying to minimize bubble entrapment. Lingering bubbles can be manually ruptured using a needle.
Cure the cast phantom at room temperature for at least 24 hours. A container may be used to ensure dust does not settle on the phantom while curing. To perform demolding, dissolve the ABS by submerging the phantom in acetone.
Sonicate for at least 15 minutes using powers up to 70 watts. Thoroughly rinse the phantom with isopropyl alcohol and then deionized water to remove solvent residues. Using an optical microscope with an attached camera in image capture software, capture an image of a critical feature within the phantom under a magnification that maximizes the feature within the field of view.
Capture an image of an appropriate calibration reticle at the same magnification. Load both images into ImageJ by dragging them onto the toolbar. Click on the calibration reticle image to make it active and then select the line tool.
Using the mouse, draw a line along a feature of a known distance and select analyze. Set scale from the ImageJ menu. Enter the length of the feature in the field labeled known distance and its unit in the field labeled unit of length.
Check the box labeled global to apply this calibration factor to all open images. Make the image of the phantom critical feature active and use the line tool to draw a line along a feature of interest. From the ImageJ menu select analyze, measure to measure the length of the line.
Compare the expected value against the value in the column marked length in the results window to confirm phantom fidelity. To make the mock blood solution mix deionized water and glycerol in a 60 to 40 ratio by volume. Add one milliliter of 2.5%fluorescent polystyrene bead solution to the mock blood solution, then homogenize the mixture on a magnetic stir plate at 400 rpm for 10 minutes.
Perform the in vitro circulatory system setup as described in the text protocol. Determine the calibration ratio for the video imaging as before. An acrylic sheet may be placed over the microscope stage before placing the PDMS phantom to protect the microscope from unintentional spills.
To setup the apparatus, place the PDMS phantom on the stage of the fluorescence microscope. Connect the phantom to the gear pump and introduce the mock blood solution. Set the pump motor controller for the desired flow rate based on the pump calibration curve.
Run the pump for one to five minutes prior to the experiment to ensure steady state conditions. If bead sticking is observed after an experiment, sonicate the phantom in an aqueous detergent solution using powers up to 70 watts. Cleanliness of the model is also critical for vector field fidelity as beads stuck to the surface of the phantom will result in interrogation windows with zero displacement.
To perform image processing, drag the save AVI file onto the ImageJ window to import it. Select the box marked convert to gray scale. From the ImageJ menu select analyze, generate histogram to generate a histogram of image pixel intensities.
Take note of the mean and standard deviation for the unprocessed image. From the ImageJ menu select image, adjust brightness and contrast to apply a brightness contrast filter. On the brightness and contrast menu, click the set button to define the image limits.
Set the minimum value to be the mean value plus one standard deviation and the maximum value to be the maximum intensity of the image. From the ImageJ menu select process, noise, despeckle to reduce the number of saturated pixels. Then select process, filters, gaussian blur with a radius of 1.5.
This will reduce artifacts arising from the occasional removal of illuminated pixels in a 3x3 neighborhood by the prior despeckling operation. Click on the polygon tool and then click on the image to outline the region of interest. From the ImageJ menu select edit, clear outside to remove sensor noise in locations where no signal is expected which can decrease the overall signal to noise ratio.
Proceed to data analysis as describe in the text protocol. Shown here is the completed phantom highlighted with dimensions as well as the particle image velocimetry or PIV region of interest. This figure shows image intensity contour plots and surface plots resulting from fluorescent beads in the perforator artery during video capture.
This demonstrates the improvement in signal to noise ratio after intensity capping is performed. Shown here are the resultant vector fields obtained on raw video, after intensity capping and then again after the intensity capped vector field is validated using the normalized median test. As post processing and vector validation techniques are employed the vector field becomes more uniform and more closely resembles the expected profile for flow in a circular channel.
While attempting this procedure it's important to focus on minimizing the noise in the acquired signal itself. While this protocol outlines software based techniques to mitigate these aberrations, care should be taken at each step to reduce their occurrence. Don't forget that working with acetone can be extremely hazardous and you should always wear personal protective equipment as well as work under a fume hood away from any ignition sources while performing this procedure.
