list to generate lineage trees (Fig. 3b). Once the segmentation of different cells in the movie is completed, all the
parameters for each individual cell at each time frame can
be retrieved simultaneously by pressing “Measure” (see
Note 36). The measurements can be exported as .csv
files and used to plot growth rates and fluorescence as a
function of time and/or genetic relatedness (see
Note 37).
(b) Plot profile: This macro can be used to analyze the cellular
patterns of protein localization. Select the “Segmented
Line” and adjust the line width according to the cell
width. Draw the segmented line throughout the cell
length and call the function “Plot Profile,” which will
generate a 2D plot of the fluorescence intensity (y-axis)
along the cell length (x-axis). Press “List” to get the
numeric values, which can be exported as .csv files and
used to generate, for instance, heat maps of fluorescence
localization.
(c) Cell counting: This plug-in can be used to score different
cell types or events occurring over time, such as division
and death events. Open the “Cell Counter” plug-in and
“Initialize” the image stack to be analyzed. “Add/Remove
Counters” to refer to specific events. Select a given
“Counter Type” and click on the cells experiencing that
specific event, using the “Point” selection tool. Score
multiple event types over time, and then, press “Results”
to get the final count of all events for each time frame.
Save the file containing the ensemble of scored events over
time as .csv file.
4. The measurements stored within .csv files can be finally
organized for further processing, plotting, and statistical analysis in other programs such as Microsoft Excel or GraphPad
Prism, or using open-source programming languages and
tools, such as RStudio or Python.
4 Notes
1. Tween 80 (0.05%, final concentration) can also be used, but is
not recommended due to its instability.
2. 7H9 is supplemented with ADS (0.5% BSA, 0.2% dextrose,
0.085% NaCl) and can be replaced with any other growth
medium compatible with mycobacteria, such as Sauton,
Dubos, M9 minimal medium, etc. Minimal media are particularly attractive for fluorescence imaging, thanks to their low
auto-fluorescence. In contrast, rich media tend to quench
fluorescence.
222
Giulia Manina and Neeraj Dhar
parameters for each individual cell at each time frame can
be retrieved simultaneously by pressing “Measure” (see
Note 36). The measurements can be exported as .csv
files and used to plot growth rates and fluorescence as a
function of time and/or genetic relatedness (see
Note 37).
(b) Plot profile: This macro can be used to analyze the cellular
patterns of protein localization. Select the “Segmented
Line” and adjust the line width according to the cell
width. Draw the segmented line throughout the cell
length and call the function “Plot Profile,” which will
generate a 2D plot of the fluorescence intensity (y-axis)
along the cell length (x-axis). Press “List” to get the
numeric values, which can be exported as .csv files and
used to generate, for instance, heat maps of fluorescence
localization.
(c) Cell counting: This plug-in can be used to score different
cell types or events occurring over time, such as division
and death events. Open the “Cell Counter” plug-in and
“Initialize” the image stack to be analyzed. “Add/Remove
Counters” to refer to specific events. Select a given
“Counter Type” and click on the cells experiencing that
specific event, using the “Point” selection tool. Score
multiple event types over time, and then, press “Results”
to get the final count of all events for each time frame.
Save the file containing the ensemble of scored events over
time as .csv file.
4. The measurements stored within .csv files can be finally
organized for further processing, plotting, and statistical analysis in other programs such as Microsoft Excel or GraphPad
Prism, or using open-source programming languages and
tools, such as RStudio or Python.
4 Notes
1. Tween 80 (0.05%, final concentration) can also be used, but is
not recommended due to its instability.
2. 7H9 is supplemented with ADS (0.5% BSA, 0.2% dextrose,
0.085% NaCl) and can be replaced with any other growth
medium compatible with mycobacteria, such as Sauton,
Dubos, M9 minimal medium, etc. Minimal media are particularly attractive for fluorescence imaging, thanks to their low
auto-fluorescence. In contrast, rich media tend to quench
fluorescence.
222
Giulia Manina and Neeraj Dhar