Visualization and post processing tools

Visualization of data and interactive post processing

Initialize

superSeggerViewerGui can be used to visualize the data after they have been segmented, and create plots, histograms, and other analysis figures. In order to load the GUI version, type superSeggerViewerGui in the console window. Start then by selecting an image directory by pressing the folder icon at the top left (). This will open something similar to this:

You can then zoom in (), zoom out (), pan (), and save () the image by using the respective icons at the top left.

The GUI is divided into five panels: Main menu, display options, link options, output options, and clist/gate options. In the following, I will go through all the functionalities within each of these panels.

Main menu

Here you can go to the previous or the next frame. This is also possible by using the left and the right arrow key (make sure your mouse is an arrow), respectively. You can also type in a specific frame number that you want to display. The image directory shows the current folder presented which was set initially, and can be changed by using the folder icon () at the top left. The channel is 0 for phase images and 1, 2, 3, etc. for fluorescence images. Finally, the xy directory corresponds to the xy directory presented.

Display options

• Find cell #: Marks with a yellow X on the screen where the cell with the id number # is located.

• IDs: Displays the ID numbers of the cells on the image (shown above in panel A).

• Regions: Displays the ID numbers of the regions instead of the cells on the image.

• Cell fill: Displays filled cell regions. The different colors indicate the different states/errors of the cells (shown above in panel A).

  • Blue: Cells where both birth and division are observed (stat0 = 2).
  • Turquoise: Cells born from a good division but did not end to a good division (stat0 = 1).
  • Green: Cells of which a birth was not observed (strays or first frame cells).
  • Purple: Cells that had errors during linking.
  • Red outline: Cels which will divide on the next frame or have just divided.

• Cell outline: Shows the outlines of all the cells.

• Phase: Displays the phase image when checked, and the mask when not checked.

• Phase edit box : 0 - 1 weight of phase/mask image.

• Cell poles: Old poles are marked with an ‘o’, new poles with a ‘*’, and the sisters are linked with a line from their centroids.

• Complete cell cycles: Uses only cells where a complete cell cycle was observed (both birth and division) in the post-processing calculations. It also creates a gate that gates for full cell cycle (stat0 = 2).

• Legend: displays a legend for the poles and cell fill.

• Region scores: Shows region scores. Scores are from 50 to -50, with 50 indicating a good region/cell.

• Use seg files: Uses the initial segmentation frame files, which are made before linking instead of the final frame files (error files). It shows region numbers instead of cells.

• False color: Displays fluorescence in false color (shown above in panel F).

• Filtered fluor: Filters the background fluorescence in the image shown (shown above in panel C).

• Foci scores: Shows the score of the foci fit to fluorescence (shown above in panel B and C).

• Log view: Shows log of fluorescence to reduce variation at high intensity.

• Composite: displays a composite image with all fluorescence channels found. Uses the color order set in the constants (CONST.view.fluorcolor).

##Link options

• Show linking: Displays lines from centroid to centroid from this frame to the previous and next frame.
• Show daughters: Shows the daughters in the linking.
• Show mothers: shows the mother in the linking.

Edit segments:

Opens another GUI that will allow you to turn on and off segments. In red are the true segments and in blue the false segments. The fixed segments are gray. More info here.

Edit links:

Opens another GUI that allows to change the links between cells. More info on how to use it here.

Output options

Double click on the options to view the result. Some of the options may not appear if you did not run segmentation all the way through.

• Save output as .mat: When ticked, the output options mentioned below will save a .mat file.
• Max cells: Maximum number of cells used for the output options.
• Cell # : Number used to display the cell kymograph, move, tower and lineage.
• Cell Kymo Calculates and shows a kymograph for the cell with the number # (shown in panel C above).

• Cell Movie: Creates a movie for fluorescence channel for the cell with id # for the duration of its lifetime. It gives you the option to save the movie at the end.
• Cell tower: Shows a rotated cell tower for the cell with the number # (shown in panel B above).

• Kymograph mosaic: Calculates and shows a mosaic of separate kymographs for all cells in the current xy position. (Limit of min( Max cells or 100 cells))

• Cell tower mosaic: Calculates and shows a mosaic of separate towers for all cells in the current xy position. (Limit of min( Max cells or 100 cells))

• Consensus kymograph: Calculates and shows the consensus kymograph for the current xy position. (Limit of min (Max cells or 1000 cells)). It creates an imArray that is used for future displays of consensus, so that it is not calculated again.

• Consensus: Calculates and shows the consensus image for the current xy position. (Limit of min (1000 cells, CONST.view.maxNumCell)) (Shown in panel A above). It creates an imArray that is used for future displays of consensus, so that it is not calculated again.

• Make field movie: Creates a movie for all time frames for the specific xy position using the visualization settings you have for superSeggerViewer. It will give you the option to choose the start, skip and end frame of the movie. It gives you the option to save the movie at the end.

• Make field mosaic: Creates a mosaic for all time frames for the specific xy position using the visualization settings you have for superSeggerViewer. It will give you the option to choose the start, skip and end frame of the movie. (Shown in panel E above).

• Lineage: It displays a lineage tree for cells with more than 3 cells in their tree. It displays the trees for the cells in cell# or for all the cells if cell # is empty. It also displays the number of cells with the time frame and when the first error appears. (Shown in panel I above).

• Cell Info: Clicking on the Cell Info button allows you to click on a cell in the frame. In the console different cytometry and fluorescence, information will be displayed about that cell in this specific frame, such as cell id, area, pole orientation, cell length and width, pole age, fluorescence statistics etc.

*Outline figure: Make a publication-quality figure with vector cell outlines.

Clist/Gate options

• Histogram: Displays a histogram for a quantity in the clist. (Shown in panel F above).
• Gate: Creates a gate on a quantity of your choice.
• TimeClist: Displays how the cells change in time (frame). (Shown in panel H above).
• Move gated cells: Moves cell files that are not within the gated limits to a separate folder.
• Plot two clists: Opens a dialog box where you can select two quantities in the clist. This will then display a bivariate histogram (dot plot) of these two quantities. (Shown in panel G above).
• Clist for all xy: It creates a combined clist for all xy positions and saves it in the main directory as clist_comp.mat.
• Clear gates: Deletes all gates made. If there is a number to the edit box next to it, it clears that specific gate.
• Exclude ids (from img): Excludes / Gates a space-separated list of cell ID numbers in the outputs and image outlines. You can either type in the cell ID numbers that you want to exclude or you can select them by activating the ‘from img’ and then pressing on the image.
• Include ids (from img): Only the cells with ID numbers in the given space separated list are included in the outputs and outlined in the image. You can either type in the cell ID numbers that you want to include or you can select them by activating the ‘from img’ and then pressing on the image.

**For additional clist and gating options, the gateToolGui offers more extensive options. **

Constants viewing options

Some of the parameters in the constants can be modified to change how things are viewed in the SuperSeggerViewer. In order to change those, you need to modify directly the constants in your image directory. Directions to do that are found here. Some values that you may want to modify are :

• CONST.getLocusTracks.FLUOR1_MIN_SCORE = 3; Only fluorescence 1 (c2) foci above this score are shown
• CONST.getLocusTracks.FLUOR2_MIN_SCORE = 3; etc..
• CONST.view.fluorColor = {'g','r','b','c','o','y'}; The ordering of colors for fluorescence channels
• CONST.view.maxNumCell = 1000; Maximum number of cells used for consensus images and mosaics.