Plotting Temporal Localization Part 2 - veeninglab/BactMAP GitHub Wiki

Import the data

To make the kymographs, cell towers and trajectory plots, we need the following data:

  • TIFF files of the original time-lapse movie of the fluorescence channels with DnaX-GFP (X.tif) & FtsZ-RFP (FTSZ.tif).
  • the results of cell segmentation/tracking. I used Oufti to segment the cells, the results are in 20stl.MAT.
  • the results of fluorescence tracking (both channels). I tracked the fluorescence with ISBatch’s Single Molecule Biophysics plugin. The results are saved in results_ftsztraj.csv and Resultstraj_dnax.csv.

Find more information on the gathering of the data and the image analysis here.

The TIF files

To import the two TIF files, use BactMAP’s function extr_OriginalStack(). This function takes the x and y coordinates of each pixel, together with the intensity value and turns them into dataframes, one per image.

FTSZ <- extr_OriginalStack("Fig5A-C/FTSZ.TIF")
DNAX <- extr_OriginalStack("Fig5A-C/X.TIF")

The cell outlines

To import the cell outlines I tracked using Oufti, use extr_Oufti(). This function takes the matlab or csv output of Oufti and returns a list of dataframes, including the cellList (a clone of the csv oufti output) and the mesh (a dataframe describing the cell outlines & dimensions). For plotting the cell shape and internal localization over time, I need the mesh dataframe, so I will call for that one directly by putting $mesh behind my call.

extr_Oufti() takes a .mat or .csv file from Oufti and the argument mag. mag is the magnification converter, or the pixel size of the microscopy image. I saved a few of my most used converters in BactMAP, you can access them with the function getPixels2um(). Use the function addPixels2um() to add a new conversion factor to the list.

myMesh <- extr_Oufti("Fig5A-C/20stl.mat", mag="100x_DVMolgen")$mesh

The tracked fluorescence

I tracked the fluorescent spots & filaments using the Peak Fitter & Particle Tracker of the Single Molecule Biophysics ImageJ Plugin from ISBatch.

To import the tracks, use the function extr_ISBatch(). This function returns a list of dataframes, including the cellList (a clone of the csv output of the Peak Fitter) and the spotframe (a list containing spot identifiers & coordinates). Since I only need the spotframe, I subset this one by putting $spotframe behind my call.

tracks_FtsZ <- extr_ISBatch("Fig5A-C/results_ftsztraj.csv")$spotframe

tracks_DnaX <- extr_ISBatch("Fig5A-C/Resultstraj_dnax.csv")$spotframe

Looking at your imported data

I have five datasets loaded into my environment: DNAX, FTSZ, myMesh, tracks_DnaX and tracks_FtsZ. Explore them using the commands summary() and View() (see part 3 of the Segmentation Tutorial for examples), and check the data structure documentation of the mesh dataframe, spot dataframe and imported image lists.**

:arrow_left: Plotting Temporal Localization Part 1: Before Getting Started Plotting Temporal Localization Part 3: Making Kymographs :arrow_right: