9. Different microscope vendors, such as Cytiva, Leica, Nikon,
Olympus, PerkinElmer, and Zeiss, provide integrated microscope setups suited for live cell microscopy. Here, we exclusively list (in brackets) the setup that we use in our labs.
10. Besides ImageJ, there are several software packages (freely
available) which can be adapted for analysis of image sequences,
such as CellProfiler [46], Fiji [47], Icy [48], MicrobeJ [49],
ImJoy [50], and BactMAP [51].
11. To generate bacterial fluorescent reporter strains, either promoter or protein fusions, it is important to choose suitable
fluorescent proteins (FPs), which are bright, have a fast maturation time, are photostable, and excitation of which does not
harm the cells. Proteins such as EGFP, sfGFP, mCitrine,
dsRed2, Tomato, mcherry, mKate2 work successfully in mycobacteria [22, 25–32]. Importantly, mycobacteria exhibit autofluorescence when excited at 405 nm, so this has limited the
use of cyan FPs [52]. It is essential to carry out control experiments to verify that exposure to fluorescent excitation is not
affecting the growth rate or behavior of mycobacteria. To
generate dual-fluorescent reporter strains, combined FPs
must have minimal spectral overlap.
12. When culturing M. tuberculosis, put the PETG bottle in a
secondary air-tight container before placing it in the shaker.
13. Use freshly prepared high-purity low-melt agarose.
14. For quick snapshot microscopy, mycobacteria can also be
imaged directly without using an agarose pad. Place 0.6 μL of
culture between two glass coverslip slides (24 Â 50 mm). In
case of M. tuberculosis, a smaller-sized coverslip can be used on
top and the edges sealed with fast-drying glue. To insure that
bacilli are static during imaging, rub the coverslip above on the
coverslip below, without applying too much pressure with the
fingertip, until the two coverslips no longer slide. This operation must be done with extreme caution, to avoid both breaking the slides and crushing the cells. When working with
M. tuberculosis, handle the coverslips with great care, as they
are very fragile. Transfer the slide from the biosafety cabinet to
the microscope in a secondary container.
15. The agarose pad should be flat and without bubbles. Try to
avoid over-drying the agarose pads, as this will cause irregularities on the surface. The agarose pads can be stored at 4
C just
for a few h.
16. Use a scalpel to transfer the agarose pads. Avoid damaging the
surfaces, as this will affect the seeding as well as imaging of
bacteria.
224
Giulia Manina and Neeraj Dhar
Olympus, PerkinElmer, and Zeiss, provide integrated microscope setups suited for live cell microscopy. Here, we exclusively list (in brackets) the setup that we use in our labs.
10. Besides ImageJ, there are several software packages (freely
available) which can be adapted for analysis of image sequences,
such as CellProfiler [46], Fiji [47], Icy [48], MicrobeJ [49],
ImJoy [50], and BactMAP [51].
11. To generate bacterial fluorescent reporter strains, either promoter or protein fusions, it is important to choose suitable
fluorescent proteins (FPs), which are bright, have a fast maturation time, are photostable, and excitation of which does not
harm the cells. Proteins such as EGFP, sfGFP, mCitrine,
dsRed2, Tomato, mcherry, mKate2 work successfully in mycobacteria [22, 25–32]. Importantly, mycobacteria exhibit autofluorescence when excited at 405 nm, so this has limited the
use of cyan FPs [52]. It is essential to carry out control experiments to verify that exposure to fluorescent excitation is not
affecting the growth rate or behavior of mycobacteria. To
generate dual-fluorescent reporter strains, combined FPs
must have minimal spectral overlap.
12. When culturing M. tuberculosis, put the PETG bottle in a
secondary air-tight container before placing it in the shaker.
13. Use freshly prepared high-purity low-melt agarose.
14. For quick snapshot microscopy, mycobacteria can also be
imaged directly without using an agarose pad. Place 0.6 μL of
culture between two glass coverslip slides (24 Â 50 mm). In
case of M. tuberculosis, a smaller-sized coverslip can be used on
top and the edges sealed with fast-drying glue. To insure that
bacilli are static during imaging, rub the coverslip above on the
coverslip below, without applying too much pressure with the
fingertip, until the two coverslips no longer slide. This operation must be done with extreme caution, to avoid both breaking the slides and crushing the cells. When working with
M. tuberculosis, handle the coverslips with great care, as they
are very fragile. Transfer the slide from the biosafety cabinet to
the microscope in a secondary container.
15. The agarose pad should be flat and without bubbles. Try to
avoid over-drying the agarose pads, as this will cause irregularities on the surface. The agarose pads can be stored at 4
C just
for a few h.
16. Use a scalpel to transfer the agarose pads. Avoid damaging the
surfaces, as this will affect the seeding as well as imaging of
bacteria.
224
Giulia Manina and Neeraj Dhar