27. Do not over tighten the screws, as this might cause the coverslip to crack. Do not keep them too loose, as this might cause
leakage.
28. Make sure that the device assembly is clamped firmly to the
stage. Because the imaging is done using a 100Â objective,
even small drifts can cause loss of the chosen imaging points
and focusing.
29. Check that there are no leakages around the device and that the
medium drops from both outlet tubings. To accelerate the
arrival of the liquid into the device, increase the perfusion
rate temporarily to 25 μL/min; however, avoid pushing the
syringe plunger manually. When switching the medium type
and syringe, prefill the female Luer connectors with the new
medium, to prevent the presence of air in the microfluidic
system.
30. When working with M. tuberculosis, autoclave the device before
sonication.
31. Switch on the environmental chamber at least 24 h before
using the microscope and allow all the microscope components
(stage, immersion oil, environmental chamber) to stabilize to
the correct temperature. Small differences in temperature can
cause drifting of the device as well as focusing issues.
32. When choosing the fields of view for time-lapse imaging, avoid
points that are too close to one another, to minimize photobleaching and phototoxicity. Also, avoid areas that are overcrowded with bacteria if any. On the other hand, it is critical to
follow a sequential trajectory, which starts and stops in the
same area, and to select points that are not too far apart from
each other, to avoid autofocus issues and loss of immersion oil
during movement of the stage. To generate this trajectory, it is
also needed to select a few blank points to walk through the six
areas of the device and to make sure that the objective lens is
always in contact with the coverslip and never collides with the
aluminum frame.
33. The frequency of imaging is dependent on the exposure conditions. To minimize phototoxicity, decrease the exposure
times when imaging multiple fluorescent channels or image
the points less frequently. Mycobacteria growing on minimal
medium are more sensitive to phototoxicity.
34. Software-based autofocus in conjunction with hardware-based
autofocus systems offers the best and most robust solution of
image-based autofocusing for mycobacteria. Most microscope
vendors now provide hardware-based autofocus systems, such
as Olympus’s Z-Drift Compensator, Nikon’s Perfect Focus
System, Zeiss’s Definite Focus, Leica’s Adaptive Focus Control, and Cytiva’s UltimateFocus Module, which is a focus
assist unit based on a 785 nm laser.
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Giulia Manina and Neeraj Dhar
leakage.
28. Make sure that the device assembly is clamped firmly to the
stage. Because the imaging is done using a 100Â objective,
even small drifts can cause loss of the chosen imaging points
and focusing.
29. Check that there are no leakages around the device and that the
medium drops from both outlet tubings. To accelerate the
arrival of the liquid into the device, increase the perfusion
rate temporarily to 25 μL/min; however, avoid pushing the
syringe plunger manually. When switching the medium type
and syringe, prefill the female Luer connectors with the new
medium, to prevent the presence of air in the microfluidic
system.
30. When working with M. tuberculosis, autoclave the device before
sonication.
31. Switch on the environmental chamber at least 24 h before
using the microscope and allow all the microscope components
(stage, immersion oil, environmental chamber) to stabilize to
the correct temperature. Small differences in temperature can
cause drifting of the device as well as focusing issues.
32. When choosing the fields of view for time-lapse imaging, avoid
points that are too close to one another, to minimize photobleaching and phototoxicity. Also, avoid areas that are overcrowded with bacteria if any. On the other hand, it is critical to
follow a sequential trajectory, which starts and stops in the
same area, and to select points that are not too far apart from
each other, to avoid autofocus issues and loss of immersion oil
during movement of the stage. To generate this trajectory, it is
also needed to select a few blank points to walk through the six
areas of the device and to make sure that the objective lens is
always in contact with the coverslip and never collides with the
aluminum frame.
33. The frequency of imaging is dependent on the exposure conditions. To minimize phototoxicity, decrease the exposure
times when imaging multiple fluorescent channels or image
the points less frequently. Mycobacteria growing on minimal
medium are more sensitive to phototoxicity.
34. Software-based autofocus in conjunction with hardware-based
autofocus systems offers the best and most robust solution of
image-based autofocusing for mycobacteria. Most microscope
vendors now provide hardware-based autofocus systems, such
as Olympus’s Z-Drift Compensator, Nikon’s Perfect Focus
System, Zeiss’s Definite Focus, Leica’s Adaptive Focus Control, and Cytiva’s UltimateFocus Module, which is a focus
assist unit based on a 785 nm laser.
226
Giulia Manina and Neeraj Dhar