Image-Guided Robotic Cell Microinjection: An Automated High-Throughput Technique for the Precise Delivery of Fluorescently-Labeled Polysaccharides Into Single Neuronal Cells in Organotypic Brain Slice Cultures
Transcript
Turn on the computer, microscope, microscope camera, manipulators, pressure rig, and pressure sensor. Load the application by clicking the file "launchapp.py" in the main folder downloaded from GitHub, and specify the device settings in the pop-up screen.
To prevent unwanted clogging, create an outward pressure before submerging the pipette into the solution. Slide the 'Compensation pressure' bar to 24% to 45% and click 'Set values.' Next, tune the pressure by turning the mechanical pressure valve knob to 1 to 2 PSI, as indicated by the pressure sensor. Transfer the slices to the center of a 3.5-centimeter Petri dish containing 2 milliliters of pre-warmed CIMM.
Then, place the Petri dish on the microinjection stage that has been preheated to 37 degrees Celsius. Load the microinjection pipette with 1.4 to 1.6 microliters of microinjection solution using a long-tip plastic pipette, and insert the microinjection pipette into the pipette holder.
Using the lowest magnification on the microscope, bring the slice into focus, and guide the micropipette to this field of view so that it is focused on the same plane as the slice target. Switch the output of the microscope to the camera to see the FOV and the application.
Click the magnification button in the top left of the interface to initiate device calibration. When a window prompts to select the magnification, select the '10x' and press 'OK.' The software assumes the internal objective lens is 10x. Refocus the pipette tip using the micrometric wheel of the microscope, and click the pipette tip with the cursor.
Next, press the 'step 1.1' button and press 'OK' in the pop-up window. The pipette will move in the Y direction. Click the tip of the pipette and press the 'step 1.2' button. Lastly, enter '45' in the 'Pipette Angle' box and press the 'Set Angle.' Enter desired parameters into the 'Automated Microinjection Controls' panel and set the speed to '100%.' When finished, click 'Set Values.'
Click the 'Draw Edge' button and drag the cursor along the desired trajectory in the pop-up window to define the trajectory of injection. For micro-injecting neurons, target the basal side of the telencephalon. Bring the pipette to the start of the line and click its tip. Then, click 'Run trajectory' to start micro-injecting. Repeat this step for every plane of injection targeted.
