- ONIX Microfluidic Control Box
- ONIX Y04C plate
- Set up microscopy systems
- Turn on systems associated with microscopy to allow time for device equilibration.
- Open MetaMorph software.
- Switch on the ONIX control box.
- Prepare the microfluidics plate
- The plate is shipped with PBS in the wells to prevent the channels from drying out. Pipette & discard the PBS from wells in the channels you'll use for your experiment.
- Add 50-100uL of the media you plan to use in columns 1-6 and the cell loading column.
- Ensure the ONIX control box "Ready" indicator is lit. Vacuum seal the plate by seating the manifold over the plate and switching the vacuum on from the ONIX control box. You may need to apply light pressure to the manifold to help establish the seal.
- Once the "Vacuum" indicator has lit, the manifold is sealed to the plate. Open the ONIX control software.
- Select the model of plate. (Yeast → Y04c)
- Prime the channel by turning on lanes 1-6 at PSI 8. This serves to flush remaining PBS from the reservoirs and cell chamber. Run for 2-3 minutes.
- Repeat the priming process on the cell loading column at PSI 6.
- Release the seal by turning the vacuum switch off. Remove the manifold.
- Replace fresh media in the reservoirs in a quantity appropriate for your experiment, not to exceed 250uL. (You may assume roughly 15 uL/hr of flow at PSI 6 with a chamber refresh time of roughly 1 minute.)
- All reservoirs should contain minimum 50uL fluid to prevent reservoirs from drying out.
- Prepare your cells for loading into the microfluidic device. 100uL of OD 0.01 cells should be sufficient.
- Prepare for microscopy and load cells into the flow chamber
- Place the microfluidic plate in the appropriate tray for the microscope stage. Secure the plate using 2 strips of tape, being careful not to obscure the light path or tilt the microfluidic plate. Note that the tape should not wrap around to the underside of the stage tray, as this will cause the tray to lay unevenly. The suggested method is to carefully trim the tape by cutting with scissors along the edge of the tray.
- Apply a generous drop of immersion oil to the objective.
- Secure the plate on the stage. Ensure there is sufficient slack in the manifold tubing to prevent torque being applied during the course of the experiment.
- Establish Kohler illumination and proper focus depth. The channel position indicators will be useful for this.
- Before loading cells but after setting focus, find an empty segment of the chamber and take background and shading reference images. See below for details.
- To load cells, open valve 8 at PSI 6. Once cells begin to appear, short pulses of a few seconds each should be used until the desired cell density is reached in each channel; it is recommended to open and close the valve manually rather than using the "Load Cells" button. Note that cells are likely to collect around the chamber edges (particularly near the chamber outlet) and at the leading edge of chamber position indicator marks. Increasing the loading pressure may help in trapping larger cells in the chamber.
- Taking Background/Shading reference images
- Open the Acquire menu's "Correction" tab
- For the background reference, "Acquire Background" with the shutter closed and lights off. Save as "CFPbkgd.tif" in your experiment's directory.
- For the shading reference, acquire and display the image. Process the image by applying a Morphological Filter; you want to use a Lomo filter with a 16px diameter circle. Select the sequential processing checkbox; do not use reconstruction. Save as "CFPshading.tif"
- Repeat for the other fluorescence channels.
Metamorph MDA Setup
Open the Metamorph Multi Dimensional Acquisition menu from the "Apps" dropdown menu.
- Main tab
- Check Timelapse
- Check Multiple stage positions
- Check Multiple wavelengths
- Check Z series
- Uncheck Stream
- Check Run journals
- Provide a description of your experiment
- Select a target directory to save into
- Choose a base name for your experiment
- Set the interval between timepoints (recommended: 5 minutes)
- Select the length of your experiment. This will set the number of timepoints
- Add positions to image. Press "+" at each location to add it to the list (recommended # of positions: 4 per channel, 16 total)
- No Z travel offset is needed
- After selecting all positions, go through them again before imaging to check for stage drift along the z axis
- Set the number of wavelengths (recommended: 5)
- No separate binning needed
- Set exposure settings for each wavelength -- generally, 50ms, 3500 EM gain for fluorophores, no gain for BF
- Z series
- Perform Z series on one wavelength at a time
- Range: 0.4 microns recommended
- Increment: step size, 0.2 microns recommended
- Number of steps: 2 recommended
- Before each image, run MDA OOF Check Lumen to set image acquisition settings
- After each image, run MDA OOF Check After to set TL lamp
- At the start of each position, run MDA Autofocus
- Default display is appropriate
- Check the predicted image file size requirements
- Make sure your selected save location has sufficient disk space
After starting the experiment, it's recommended to watch the first two timepoints be measured to ensure the system is set up correctly.
- After running your experiment, run Format MDA Time Series and follow the prompts to prepare your data for processing in TrackGUI software
MATLAB Cell tracking