Biology Reference
In-Depth Information
transcription and translation machineries, which occupy very clear subcellular
domains during rapid growth in rich media, compared to that seen in minimal media
(
Lewis
et al.
, 2000
). Consistency is the key to obtaining clear reproducible images.
This is of particular importance when comparing fluorescently labelled proteins from
different strains. The media, the glassware and the incubation parameters all need to
be standardised in order to be able to compare with confidence how two labelled
proteins behave in different strains (
Buescher
et al.
, 2012
).
4.1
Liquid
Under most circumstances, cells being prepared for microscopy can be grown in LB
at 37
C with aeration. Microscopy of stationary phase cells can be done using cells
grown in overnight cultures with any required supplements. For microscopy of expo-
nential growing cells, overnight cultures should be diluted back to an OD
600
of 0.01
and grown with aeration to an optical density of approximately 0.3. One way to
reduce waiting time and maximise the time spent on the microscope is to culture cells
in 96-well microplates. This allows up to 12 different strains to be comfortably
imaged by using simple dilutions. Fill the wells along Row A of the microplate with
200
L of over-
night culture to the 200 mL in Row A, mix thoroughly and take 100 mL to mix with
the contents of Row B. Continue this 1:2 dilution down the plate to Row H and dis-
card the final 100
m
L of growth medium and then add 100
m
L to all other wells. Add 2
m
L. This gives eight dilutions of up to 12 cultures, providing flex-
ibility in imaging without the use of excessive glassware and incubator space.
Cells carrying pLau53 require induction at an OD
600
of
m
0.05 with 0.05% (w/v)
L
-arabinose and grown for a further 1-3 h. Cells that require lower levels of fluores-
cent protein production can be induced with 0.01% (w/v)
L
-arabinose at an OD
600
of
0.2-0.6. Variability will occur with respect to the requirements for induction and
should be optimised for each strain and circumstance.
If visualisation of the nucleoid is required, 5
L of DAPI (4
0
,6-diamidino-2-
m
phenylindole; 30 ng/mL stock) is added to 5
m
L of the culture immediately prior
to being placed on the slide.
4.2
Solid
Cells can be grown on a slide mounted with an agarose-based medium (1.2% aga-
rose, w/v, in minimal media, e.g. M9). We have found that several fusions lose their
localisation patterns when cells are attached to poly-
L
-lysine-coated slides, and con-
sequently, we routinely use agarose-based slides for all imaging of fluorescent pro-
tein fusions. Rich media such as LB are not recommended due to high levels of
autofluorescence although this artefact can be removed to some extent as described
below under image processing. A temperature-controlled stage may be required.
Other considerations include humidity, pH, oxygen, photobleaching and phototox-
icity. Time-lapse microscopy can then be used by taking successive frames at spec-
ified time intervals (e.g. 30 min).
