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- Cut microbore tubing into 48'' lengths, one per port. If a dial-a-wave device is used, 96" lines are necessary to compensate for the increased height of the linear actuators
- Remove the plunger from a 10ml syringe
- Note: For dial-a-wave devices, 25ml or 60ml syringes are used for the media reservoirs. The protocol is the same, but the larger syringes are required to reduce the impact of media depletion.
- Attach a luer stub to the syringe body
- Insert the luer stub into one end of the microbore tubing
- Insert a straight pin into the other end of the microbore tub
- Decant 10ml of sterile water into the syringe, letting it run down the side of the syringe body
- Flick the luer stub to clear any bubbles
- Let the water flow through the tube until all of the bubbles have been cleared
- Top the syringe with a square of parafilm
- Use the pin to poke a hole in the parafilm, and store the tube until it is time to load the chip
- Repeat steps 2 to 10 for remaining water syringes and media syringe(s)
- Once the cells have reached OD600 of ~0.1, transfer 10ml of cells to a 15ml conical tube. Spin at ~1500xg (3500RPM in a Sorvall SS-34 rotor) for 5 minutes.
- While the cells are spinning down, turn on the fluorescence source for the microscope so that the bulb can reach operating temperature.
- Note: During the first 5-10 minutes of on-time, the intensity of the fluorescence source will fluctuate until the bulb reaches operating temperature. Therefore, it is important to allow a sufficiently long warm up time. Once the source is on, leave the output at 0\% 0% to prevent accidental exposure when configuring the run.
- Decant and discard the supernatant. Add 10ml fresh LB media with antibiotics and resuspend the cells by vortexing briefly
- Repeat steps 2 to 10 with the cell syringe
Loading the device and starting the experiment
Flushing order for dual iDaw device
- Mount syringes to the carriers on the syringe tower and adjust the heights for loading
- Beginning with the media port and finishing with the cell port, insert the pin from the corresponding syringe into the port, making sure to insert the pin vertically without cutting into the PDMS
- After all pins have been inserted into the device, use a Kimwipe to remove the flushing solution from the top of the chip
- Gently gather the lines running into the chip and bend the lines slightly to prevent collision with the condenser. Affix the lines to the stage with tape
- Adjust the position of the stage until the trap is visible in the middle of the field of view
- Move the cell syringe up or down to modulate the speed of cells as they pass the trapping area
- Flick the cell line to force a single cell into the trapping area. Once a single cell has been trapped, stop flicking, as loading more than one cell can lead to premature trap filling
- Move the chip mount away from the objective and change to the 100x objective. Place a drop of microscopy oil on the 100x objective
- Replace the chip mount above the objective
Focus the 100x objective
- Configure and test the autofocus
- Set the exposure doses for phase contrast and each fluorescence channel
- Set the fluorescence source to 10% output
- Set the time between exposures and set the destination directory for the data file. Name the file in a descriptive way.
- Once all configuration is done, lower the cell syringe to the running height and ensure that flow across the trapping area is at the correct rate and is from the media source.
- Turn off the room lights, close the door, start the run and turn off the computer monitor.
- After the first exposure, check to ensure that the autofocus is functioning and that the exposure times are correct.
- Leave the scope to run for the desired time length (usually 6-8 hours for bacteria)
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- Open run file in Elements
- File > ND Document > Export ND Document
- Select the correct destination to export
- Select TIF files with a mono image for each channel
Room 318 Microscope Components
