Time Lapse of DnaX and FtsZ in the pneumococcus - veeninglab/BactMAP GitHub Wiki
Download dataset
Download a .zip of this collection here.
Do the tutorial
You can use this dataset to recreate figure 5 of our preprint (coming soon). Find the tutorial here.
General organization
This dataset is made up of two image analysis subsets:
- Fig 5A-C: 20s timelapse of DnaX-GFP and FtsZ-RFP
- Fig 5D-H: 2min timelapse of DnaX-GFP
Both sets contain fluorescence microscopy images (saved as 8-bit .TIFFs) and files generated by phase-contrast segmentation programs & spot detection programs and detection setting information.
Growth conditions & Microscopy settings
For Fig5A-C, Streptococcus pneumoniae D39 carrying dnaX::dnaX-GFP & ftsZ::ftsZ-RFP was grown in C+Y medium (Aprianto, R., Slager, J., Holsappel, S., & Veening, J. W. (2018). High-resolution analysis of the pneumococcal transcriptome under a wide range of infection-relevant conditions. Nucleic acids research, 46(19), 9990-10006.) until OD 0.2, after which it was re-inoculated in fresh C+Y medium and allowed to grow for one hour. The cells were immobilized on 1.2% agarose in PBS and brought to the microscope. The cells were imaged every 20 seconds for one hour in 30 degrees, using a 488 nm and a 568 nm laser in HiLo mode on a Deltavision Elite WideField microscope.
The movie used in Fig5D-H was used before in van Raaphorst, Kjos & Veening, 2017. In short, the cells carrying dnaX::dnaX-GFP were imaged at 37 degrees Celsius every 2 minutes for 3 hours and 48 minutes. The full conditions & microscopy settings can be found in the materials & methods of the paper.
Segmentation & spot detection
Segmentation and spot detection in Fig 5A-C
Segmentation using Oufti
For the 20s-interval time-lapse, the cells were segmented using Oufti. The specific settings can be found in the matlab file 20stl.mat. The cells were manually adjusted during the time-lapse to make sure all cells were segmented.
Spot detection using iSBatch
The fluorescent spots were detected using the Peak Fitter & Particle Tracker options of the Single Molecule Biophysics Plugin of iSBatch using standard settings.
Segmentation and spot detection in Fig 5D-H
The 2 minute-interval movie was segmented using SuperSegger. The
parameters (standard 60xBay) can be found in the file CONST.mat.
After segmentation, few manual changes to the septa were
made.
Overview of files
Files used for Fig 5A-C
| File | Type | Description |
|---|---|---|
| 20stl | .MAT | Output from Oufti segmentation of time-lapse movie |
| results_ftsztraj | .csv | Results output from particle tracking of FtsZ filaments |
| Resultstraj_dnax | .csv | Results output from particle tracking of DnaX filaments |
| FTSZ | .TIFF | 8-bit TIFF stack of microscopy of the RFP channel |
| X | .TIFF | 8-bit TIFF stack of microscopy of the GFP channel |
Files used for Fig 5D-H
| File | Type | Description |
|---|---|---|
| supseg | folder | folder containing SuperSegger output |
| supseg/CONST | .MAT | parameter settings for segmentation |
| supseg/xy1/cell | folder | folder containing .MAT files with cell mask & dimensions of each cell |
| supseg/xy1/clist | .MAT | cell dimensions & growth/division information |
| classification_eye | .csv | cells discarded because of segmentation defects or lack of fluorescence, determined by eye |
| DnaX-GFP | .TIFF | 8-bit TIFF stack of microscopy of the GFP channel |
| POL_aligned | .TIFF | 8-bit TIFF stack of phase contrast channel |