The scope of our research is directed towards lifestyle mechanobiology, specifically the behavior of cells where mechanical stress is applied. We are investigating the intracellular signaling in single cells. The proposed FMPA system utilizes fluorescence imaging combined with mechanical stimuli.
That is the aspiration pressure. There are currently further advancements that have attempted to combine more than two techniques with the microaspiration setup like microfluidics or image analysis software. Current experimental challenges lie with the fact that the setup is quite labor intensive and is operated dependent.
As the system is manually operated, there are inconsistencies that arise in specific steps, for example, filament preheating. The findings of research demonstrated there was a corresponding increase in the influx of calcium ions into the RBCs when the pressure was incrementally increased between 10 millimeter mercury to 40 millimeter mercury. This suggests that RBCs poses the ability to sense changes in their mechanical environment and respond by rapid calcium related channel activities.
This study places particular emphasis on the application of FMPA as a crucial tool for unveiling the nuanced mechanized sensitive responses showcased by RBCs under varying stimuli. To begin, mount a borosilicate capillary tube onto a P1000 micropipette polar. Use the preset program with heat set to 516, velocity set to 75, time set to 250, and pressure set to 500.
Mount one of the close ended micropipettes on a micropipette cutter to open a closed tip. Adjust the heating temperature to about 50 to 60 degrees Celsius. With a 10x eyepiece, locate the micropipette.
Use the adjustment knobs to move the micropipette close to the borosilicate glass bead. Change the eyepiece to 30x. Then move the micropipette as close to the glass bead as possible without bending the pipette tip.
Step on the heating paddle to soften the glass bead with heat. Gently insert the raw closed micropipette tip into the softened bead until the desired diameter is obtained. Release the foot pedal to let the glass bead cool down.
Then gently extract the one micrometer diameter micropipette with a clear straight cut on the closed pipette. To begin, use a diamond pencil to divide a standard glass cover slip into three equal strips. Use vacuum grease to stick a piece of the cover slip strip to the bottom of a homemade chamber holder.
Similarly, attach the second piece of the cover slip strip to the top of the chamber holder. Next, use a pipette gun to inject 200 microliters of the labeled RBC suspension between the two cover slips. To assemble the micropipette aspiration setup, mount the cell chamber onto the holder stage on a microscope platform.
Adjust the position of the chamber until it is directly below the objective. Now lower the holder until it is below the fluid level of the connected water reservoir. Inject demineralized water into the micropipette.
Then use a syringe equipped with a 34 gauge needle to remove any air bubbles. Unscrew the end of the micropipette holder halfway, and allow the water to drip from the holder for a few seconds. Next, insert the suspension filled micropipette into the holder tip and tighten the holder screw.
Place the micropipette in the cell chamber. Then locate the pipette and RBCs under the microscope. Lower the pipette tip to ensure that it is leveled with the located RBC.
Adjust the height of the water reservoir to zero the hydraulic pressure at the tip. Then slightly raise the water reservoir to generate a subtle positive pressure. Turn on the 488 nanometer fluorescent excitation light source.
Turn on the fluorescence camera and the transmitted camera. Input the exposure time, region of interest, and binning size for both cameras in the corresponding software. Then set up the acquisition number to 2000 under the multidimensional acquisition panel.
Choose the required saving directory. Next, locate the micropipette with the help of the micromanipulator. Turn on the pneumatic pressure clamp and ensure that the control box is in the external mode.
Slowly rotate the knob to compensate for any offset pressure inside the system. Launch the software that controls the pneumatic clamp. Use the electrical control panel to keep pressure controlled with a 20 millivolt per millimeter mercury conversion factor.
Now zero the pressure inside the system. Then carefully relocate the micropipette close to the RBCs. Adjust the position of the water reservoir to generate a subtle positive pressure at the micropipette tip.
Initiate acquisition in the camera software and switch on the fluorescence shutter. Type in the calculated voltage magnitude into the control panel to reach the desired pressure and aspirate an RBC. Hold the pressure for a preset period, then release it.
Similarly, repeat the experiment with the next RBC before further analysis. Incremental increase in pressure resulted in an increased mobilization of calcium ions into the RBCs.
