7. Save the edited script.
8. Manually adjust the focal plane to the bottom of the stack.
9. From the “Script” panel in Micromanager, select “Run” (see
Note 31).
For image reconstruction:
10. SRRF sequences can be processed using the Fiji plugin developed by Ricardo Henriques’ lab (https://bitbucket.org/
rhenriqueslab/nanoj-srrf/wiki/Home) [24]. The plugin
can be used to convert a raw image sequence into a single
SRRF image (Fig. 5a, b), or to process a group of SRRF
sequences. To do this, open the raw data and initialize the
plug-in by selecting “Plugins > NanoJ-SRRF > SRRF
Analysis.”
11. Select the desired SRRF reconstruction settings (see Notes 32
and 33) and click “OK” to run the script.
12. We developed a Fiji macro to reconstruct multidimensional
SRRF images (time-lapse, Z-slices) automatically. To use it,
open
the
macro
“SRRF_Processing_Timelapse
MM1.4_20190711.ijm”
(https://github.com/
RFGLabAtUofT/SRRF) in Fiji from the “Plugins > Macros
> Edit” option.
13. Edit the values for the following parameters: number of slices
per stack; number of images per SRRF reconstruction; root
directory containing the raw data (which will contain folders
time0–timeN, corresponding to time points 0 to N); output
directory to save the SRRF image reconstructions; output
directory to save average intensity projection images (for
comparison with SRRF reconstructions); and the values for
the three SRRF processing parameters (see Notes 32 and 33).
14. Save the edited script.
15. Run the script by selecting “Run” in the Fiji macro window.
Each raw burst of images will be automatically reconstructed
into individual SRRF images. SRRF images corresponding to
different Z slices of a specific time point will be saved as
independent files in a common subfolder. At the end, all the
slices corresponding to a time point will be combined and
saved as a multipage tiff file, and all the multipage tiff files
corresponding to all the movie time points will saved in the
same folder. At this point, the individual time point folders
can be deleted, and the results are opened as a hyperstack in
Fiji (Movie 5).
212
Gordana Scepanovic et al.
8. Manually adjust the focal plane to the bottom of the stack.
9. From the “Script” panel in Micromanager, select “Run” (see
Note 31).
For image reconstruction:
10. SRRF sequences can be processed using the Fiji plugin developed by Ricardo Henriques’ lab (https://bitbucket.org/
rhenriqueslab/nanoj-srrf/wiki/Home) [24]. The plugin
can be used to convert a raw image sequence into a single
SRRF image (Fig. 5a, b), or to process a group of SRRF
sequences. To do this, open the raw data and initialize the
plug-in by selecting “Plugins > NanoJ-SRRF > SRRF
Analysis.”
11. Select the desired SRRF reconstruction settings (see Notes 32
and 33) and click “OK” to run the script.
12. We developed a Fiji macro to reconstruct multidimensional
SRRF images (time-lapse, Z-slices) automatically. To use it,
open
the
macro
“SRRF_Processing_Timelapse
MM1.4_20190711.ijm”
(https://github.com/
RFGLabAtUofT/SRRF) in Fiji from the “Plugins > Macros
> Edit” option.
13. Edit the values for the following parameters: number of slices
per stack; number of images per SRRF reconstruction; root
directory containing the raw data (which will contain folders
time0–timeN, corresponding to time points 0 to N); output
directory to save the SRRF image reconstructions; output
directory to save average intensity projection images (for
comparison with SRRF reconstructions); and the values for
the three SRRF processing parameters (see Notes 32 and 33).
14. Save the edited script.
15. Run the script by selecting “Run” in the Fiji macro window.
Each raw burst of images will be automatically reconstructed
into individual SRRF images. SRRF images corresponding to
different Z slices of a specific time point will be saved as
independent files in a common subfolder. At the end, all the
slices corresponding to a time point will be combined and
saved as a multipage tiff file, and all the multipage tiff files
corresponding to all the movie time points will saved in the
same folder. At this point, the individual time point folders
can be deleted, and the results are opened as a hyperstack in
Fiji (Movie 5).
212
Gordana Scepanovic et al.