35. ImageJ or Fiji has the capability of importing most file formats
generated by different microscope vendors. Once imported,
the files can be saved as .tif sequences. These image sequences
can also be annotated in ImageJ and exported as movie files.
36. When quantifying fluorescence dynamics over time, it is important to make sure that the fluorescent lamp intensity variation is
corrected for during analysis. Background fluorescence subtraction should always be done by drawing a polygon in a
cell-free section of the field of view. It can also be useful to
have a different fluorescent protein expressed constitutively to
serve as an internal control or normalization factor.
37. In case of mycobacteria, since the events of cell division and cell
separation are temporally distinct events, it is difficult to quantify cell division events or interdivision time using phase or
cytoplasmic fluorescence imaging alone. Septation markers
such as Wag31 [30] or membrane staining using fluorescent
dyes could be used to better define these events. Dyes work
quite well with mycobacteria at concentrations spanning
between 0.5 μM and 10 μM, and 7H9 medium is compatible
with their use.
Acknowledgments
This work was supported by the Institut Pasteur and by ANR grants
(ANR-10-LABX-62-IBEID and ANR-17-CE11-0007-01) to
GM. ND acknowledges support from the Swiss South African
Joint Research Program of the Swiss National Science Foundation
(Project IZLSZ3_170912). GM & ND were supported by the
Innovative Medicines Initiative 2 Joint Undertaking (JU) under
grant agreement No 853989. The JU receives support from the
European Union’s Horizon 2020 research and innovation
programme and EFPIA and Global Alliance for TB Drug Development non profit organization, Bill & Melinda Gates Foundation,
University of Dundee.
References
1. Avery SV (2006) Microbial cell individuality
and the underlying sources of heterogeneity.
Nat Rev Microbiol 4:577–587
2. Locke JCW, Elowitz MB (2009) Using movies
to analyse gene circuit dynamics in single cells.
Nat Rev Micro 7:383–392
3. Locke JC, Young JW, Fontes M et al (2011)
Stochastic pulse regulation in bacterial stress
response. Science 334:366–369
4. Norman TM, Lord ND, Paulsson J et al (2013)
Memory and modularity in cell-fate decision
making. Nature 503:481–486
5. Smits WK, Kuipers OP, Veening J-V (2006)
Phenotypic variation in bacteria: the role of
feedback regulation. Nat Rev Microbiol
4:259–271
6. Eldar A, Elowitz MB (2010) Functional roles
for noise in genetic circuits. Nature
467:167–173
7. Garcia-Bernardo J, Dunlop MJ (2015) Noise
and low-level dynamics can coordinate multicomponent bet hedging mechanisms. Biophys
J 108:184–193
Microfluidic Microscopy of Mycobacteria
227
generated by different microscope vendors. Once imported,
the files can be saved as .tif sequences. These image sequences
can also be annotated in ImageJ and exported as movie files.
36. When quantifying fluorescence dynamics over time, it is important to make sure that the fluorescent lamp intensity variation is
corrected for during analysis. Background fluorescence subtraction should always be done by drawing a polygon in a
cell-free section of the field of view. It can also be useful to
have a different fluorescent protein expressed constitutively to
serve as an internal control or normalization factor.
37. In case of mycobacteria, since the events of cell division and cell
separation are temporally distinct events, it is difficult to quantify cell division events or interdivision time using phase or
cytoplasmic fluorescence imaging alone. Septation markers
such as Wag31 [30] or membrane staining using fluorescent
dyes could be used to better define these events. Dyes work
quite well with mycobacteria at concentrations spanning
between 0.5 μM and 10 μM, and 7H9 medium is compatible
with their use.
Acknowledgments
This work was supported by the Institut Pasteur and by ANR grants
(ANR-10-LABX-62-IBEID and ANR-17-CE11-0007-01) to
GM. ND acknowledges support from the Swiss South African
Joint Research Program of the Swiss National Science Foundation
(Project IZLSZ3_170912). GM & ND were supported by the
Innovative Medicines Initiative 2 Joint Undertaking (JU) under
grant agreement No 853989. The JU receives support from the
European Union’s Horizon 2020 research and innovation
programme and EFPIA and Global Alliance for TB Drug Development non profit organization, Bill & Melinda Gates Foundation,
University of Dundee.
References
1. Avery SV (2006) Microbial cell individuality
and the underlying sources of heterogeneity.
Nat Rev Microbiol 4:577–587
2. Locke JCW, Elowitz MB (2009) Using movies
to analyse gene circuit dynamics in single cells.
Nat Rev Micro 7:383–392
3. Locke JC, Young JW, Fontes M et al (2011)
Stochastic pulse regulation in bacterial stress
response. Science 334:366–369
4. Norman TM, Lord ND, Paulsson J et al (2013)
Memory and modularity in cell-fate decision
making. Nature 503:481–486
5. Smits WK, Kuipers OP, Veening J-V (2006)
Phenotypic variation in bacteria: the role of
feedback regulation. Nat Rev Microbiol
4:259–271
6. Eldar A, Elowitz MB (2010) Functional roles
for noise in genetic circuits. Nature
467:167–173
7. Garcia-Bernardo J, Dunlop MJ (2015) Noise
and low-level dynamics can coordinate multicomponent bet hedging mechanisms. Biophys
J 108:184–193
Microfluidic Microscopy of Mycobacteria
227