19. Ultrasonic water-bath.
20. Ultrapure water.
21. 5-mL syringes.
2.8 Time-Lapse
Microscopy Imaging
1. Inverted microscope with fluorescence capability (Delta Vision
Elite imaging system, Cytiva) (see Note 9).
2. Environmental chamber enclosing the optical components and
the stage.
3. Temperature-controlled air flow apparatus connected to the
environmental chamber.
4. High-speed
and
high-resolution
sCMOS
camera
(2560 Â 2160 pixels, pixel size 6.5 Â 6.5 μm, 15-bit, spectral
range of 370–1100 nm).
5. Motorized stage, for multipoint visiting on the x, y-axes and for
autofocusing on the z-axis (25 Â 50 mm flexure stage, Cytiva).
6. Light source for epifluorescence imaging (250 W Xenon light
source or DV Light Solid-State Illuminator 7 Colors, Cytiva).
7. Polychroic Beam Splitter suitable for different wavelengths
combinations (Cytiva).
8. Motorized shutters for single-band pass emission filters and
multicolor imaging (Cytiva).
9. Hardware-based autofocus unit, correcting for axial drift with a
50 nm precision, using a 785 nm laser (UltimateFocus Module, Cytiva).
10. 100Â oil objective (UPLFLN100XO2/PH3/1.30 NA,
0.2 mm WD, Olympus).
11. Universal condenser (IX2-LWUCD/0.55 WD ¼ 27 mm,
Olympus).
12. Phase ring (IX-PH3, Olympus).
13. Image acquisition computer: with acquisition software and
microscope controller to drive precise motion control on all
the motorized components (Workstation with softWoRx 6.0,
Cytiva).
14. Custom-built clamps, to hold the microfluidic device in position on the stage (Fig. 1d).
15. Antivibration Table (DV Advanced Isolation Table, Cytiva).
16. Image analysis computer.
2.9 Single-Cell Data
Analysis
and Processing
1. ImageJ 1.52d (Wayne Rasband, NIH, USA) (http://imagej.
nih.gov/ij/index.html) (see Note 10).
2. Open-source programming languages and tools, such as RStudio
Version 1.1.463 (https://rstudio.com/products/rstudio/).
Microfluidic Microscopy of Mycobacteria
211
20. Ultrapure water.
21. 5-mL syringes.
2.8 Time-Lapse
Microscopy Imaging
1. Inverted microscope with fluorescence capability (Delta Vision
Elite imaging system, Cytiva) (see Note 9).
2. Environmental chamber enclosing the optical components and
the stage.
3. Temperature-controlled air flow apparatus connected to the
environmental chamber.
4. High-speed
and
high-resolution
sCMOS
camera
(2560 Â 2160 pixels, pixel size 6.5 Â 6.5 μm, 15-bit, spectral
range of 370–1100 nm).
5. Motorized stage, for multipoint visiting on the x, y-axes and for
autofocusing on the z-axis (25 Â 50 mm flexure stage, Cytiva).
6. Light source for epifluorescence imaging (250 W Xenon light
source or DV Light Solid-State Illuminator 7 Colors, Cytiva).
7. Polychroic Beam Splitter suitable for different wavelengths
combinations (Cytiva).
8. Motorized shutters for single-band pass emission filters and
multicolor imaging (Cytiva).
9. Hardware-based autofocus unit, correcting for axial drift with a
50 nm precision, using a 785 nm laser (UltimateFocus Module, Cytiva).
10. 100Â oil objective (UPLFLN100XO2/PH3/1.30 NA,
0.2 mm WD, Olympus).
11. Universal condenser (IX2-LWUCD/0.55 WD ¼ 27 mm,
Olympus).
12. Phase ring (IX-PH3, Olympus).
13. Image acquisition computer: with acquisition software and
microscope controller to drive precise motion control on all
the motorized components (Workstation with softWoRx 6.0,
Cytiva).
14. Custom-built clamps, to hold the microfluidic device in position on the stage (Fig. 1d).
15. Antivibration Table (DV Advanced Isolation Table, Cytiva).
16. Image analysis computer.
2.9 Single-Cell Data
Analysis
and Processing
1. ImageJ 1.52d (Wayne Rasband, NIH, USA) (http://imagej.
nih.gov/ij/index.html) (see Note 10).
2. Open-source programming languages and tools, such as RStudio
Version 1.1.463 (https://rstudio.com/products/rstudio/).
Microfluidic Microscopy of Mycobacteria
211