23. Switch on the pump and maintain the flow rate at 10 μL/min
(see Note 29).
24. Carry out the time-lapse microscopy experiment (as in Subheading 3.8).
25. At the end of the experiment, transfer the microfluidic setup
into a biosafety cabinet and gently perfuse the microfluidic
system with 70% ethanol.
26. Transfer the device and tubings into a 250-mL beaker containing 200 mL isopropanol. Cover the beaker with aluminum foil.
27. Place the beaker in an ultrasonic water bath and sonicate the
device by for 60 min (see Note 30).
28. Discard the isopropanol and rinse the entire microfluidic system thoroughly under deionized running water and allow any
residual isopropanol to evaporate completely under a chemical
fume hood overnight.
29. Wash the tubing extensively by flowing about 50 mL of ultrapure water using a 5-mL syringe.
30. Remove the traces of water from the tubing and the device
using compressed air.
31. Wrap each tubing separately and fasten it with autoclave tape.
32. Place the microfluidic device in a 250-mL beaker keeping the
channel-side facing up.
33. Cover the beaker with aluminum foil and autoclave it. The
device is now ready for re-use.
3.8 Time-Lapse
microscopy Imaging
1. Switch on the microscope heater and set it at the required
temperature 24 h before use, to allow the temperature in the
environmental chamber to stabilize (see Note 31).
2. Observe the cells from the eyepiece using the phase channel
and record the [x, y, z] position of points in the device using the
microscope software (see Note 32).
3. Set the acquisition conditions—imaging channels; exposure
conditions; list of positions; imaging frequency; experiment
duration; autofocus settings, in the image acquisition software.
Even though these settings are variable and depend on the
fluorescence signal and experimental setup, below are listed
some conditions we use typically in our experiments when
imaging dual-fluorescent reporters in mycobacteria (Fig. 2).
(a) Imaging channels: Typically, 3 to 4 channels can be
imaged simultaneously. An example of the polychroic
beam splitters and filter sets that can be used is provided
(Cytiva). Polychroic 1 (D-F-T-C): DAPI (Ex:390/18–
Em:435/48); FITC (Ex:475/28–Em:525/48); TRITC
(Ex:542/27–Em:597/45); Cy5 (Ex:632/22–Em:679/
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Giulia Manina and Neeraj Dhar
(see Note 29).
24. Carry out the time-lapse microscopy experiment (as in Subheading 3.8).
25. At the end of the experiment, transfer the microfluidic setup
into a biosafety cabinet and gently perfuse the microfluidic
system with 70% ethanol.
26. Transfer the device and tubings into a 250-mL beaker containing 200 mL isopropanol. Cover the beaker with aluminum foil.
27. Place the beaker in an ultrasonic water bath and sonicate the
device by for 60 min (see Note 30).
28. Discard the isopropanol and rinse the entire microfluidic system thoroughly under deionized running water and allow any
residual isopropanol to evaporate completely under a chemical
fume hood overnight.
29. Wash the tubing extensively by flowing about 50 mL of ultrapure water using a 5-mL syringe.
30. Remove the traces of water from the tubing and the device
using compressed air.
31. Wrap each tubing separately and fasten it with autoclave tape.
32. Place the microfluidic device in a 250-mL beaker keeping the
channel-side facing up.
33. Cover the beaker with aluminum foil and autoclave it. The
device is now ready for re-use.
3.8 Time-Lapse
microscopy Imaging
1. Switch on the microscope heater and set it at the required
temperature 24 h before use, to allow the temperature in the
environmental chamber to stabilize (see Note 31).
2. Observe the cells from the eyepiece using the phase channel
and record the [x, y, z] position of points in the device using the
microscope software (see Note 32).
3. Set the acquisition conditions—imaging channels; exposure
conditions; list of positions; imaging frequency; experiment
duration; autofocus settings, in the image acquisition software.
Even though these settings are variable and depend on the
fluorescence signal and experimental setup, below are listed
some conditions we use typically in our experiments when
imaging dual-fluorescent reporters in mycobacteria (Fig. 2).
(a) Imaging channels: Typically, 3 to 4 channels can be
imaged simultaneously. An example of the polychroic
beam splitters and filter sets that can be used is provided
(Cytiva). Polychroic 1 (D-F-T-C): DAPI (Ex:390/18–
Em:435/48); FITC (Ex:475/28–Em:525/48); TRITC
(Ex:542/27–Em:597/45); Cy5 (Ex:632/22–Em:679/
218
Giulia Manina and Neeraj Dhar