36
- Mount the prepared slide on the epi-fluorescence microscope.
- Choose the fields with a monolayer of 100–500 cells per field
(see Note 16). - Record images at 100× magnification in phase contrast and in
fluorescence. The excitation light intensity and the exposure
time allowing detecting all fluorescent MutL foci are setup
dependent. Each experimenter needs to determine these
parameters for his system. Choose the minimum excitation
light intensity and the shortest exposure times for which all
fluorescent MutL foci are detected. This will decrease photo-
toxicity and limit fluorescence bleaching. The detection of the
fluorescent MutL foci will therefore be more accurate.
4 Notes
- In addition to bleaching the fluorescent signal, high levels of
excitation light are toxic to cells. While this is not the problem
when taking a single snapshot picture of growing cells, it is
relevant when performing time series imaging. Therefore, we
use a low level of excitation light when doing the time-lapse
imaging of the microcolonies growing on the agar pads or
when doing long-term imaging of cells growing in the micro-
fluidic chips. In these experimental setups, it is necessary to
determine experimentally the maximum excitation light level
that does not cause cell toxicity when applied with a desired
interval. This can be done by comparing, at the end of the
experiment, the fitness of the cells that were subjected to the
imaging to the fitness of the cells growing in the same setup,
but not imaged throughout the experiment. - We do not recommend adding the IPTG systematically to the
growth medium. In some media, the leaky expression is suffi-
cient and inducing the expression of yfp-mutL more will lead
to increased background cytoplasmic fluorescence. Too high
cytoplasmic fluorescence prevents the accurate detections of
fluorescent MutL foci. We found that adding the IPTG
inducer is not necessary when cells carrying Plac construct are
grown in minimal medium supplemented with casamino acids
at 0.2%. On the contrary, in LB, the leaky expression of the
fluorescent mutL from the Plac is not sufficient for comple-
menting the inactivation of the native mutL. The inducer
IPTG needs to be added to the LB growth medium at the
concentration of 0.1 mM. - For the qualitative mutagenesis test (Rif spot test) grow the
strain expressing the fluorescent MutL and deleted for the
native chromosomal mutL gene, as well as the reference wild-
3.3 Microscopy
Marina Elez et al.