All ObjectJ Examples

SporeTrackerX

15-June-2018

Norbert Vischer
Bacterial Cell Biology
University of Amsterdam

Download SporeTrackerX
Documentation (pdf)
Other links:
Nikon .nd2 Import
Embedded Macros on GIT
Shadow Directory

SporeTrackerX is an ImageJ/ObjectJ project for the analysis of germintion and outgrowth of bacterial spores.
Due to consequent usage of virtual stacks, it allows to browse through very large time-lapse data sets (TByte) that are stored on external harddisks connected to a normal desktop or laptop computer.
In addition to provide the SproreTrackerX software, the documentation (pdf) proposes a suitable file structure and explains the conversion of Nikon .nd2 to TIFF files, the analysis of bacterial germination and growth, and the usage of suitable hardware.

Important parameters are doubling time TD, start of germination time, and burst time.
SporeTrackerX is a follow-up program of SporeTracker and accepts multi-channel hyperstacks. However, only the first channel (phase-contrast) is evaluated in this example.

Features
- Handling and converting Nikon .nd2 file sets up to 1 TB.
- Preparing a file structure
- Analysis of germination and bacterial growth

Try out SporeTrackerX:

Instruction Video (4 min, 16 MB)

  1. Download and unpack DemoMovies (500 MB)
  2. Download and unpack SporeTrackerX.ojj
    (file name may include a version number)
  3. Move SporeTrackerX.ojj into the folder DemoMovies
    (the .ojj file needs to be in the movies folder)
  4. Make sure ImageJ is open and plugin ObjectJ is loaded
  5. Open SporeTrackerX.ojj by dragging it into the main window of ImageJ
    (alternatively, use menu File> Open...)
  6. "Link" the two movies to the project by dragging them into the panel for 'Linked Images'
    (Alternatively, choose menu ObjectJ>Linked Images>Link All Images from Project Folder)

Analyze:

  1. Choose menu ObjectJ>Analyze Spores
    Spores will be marked with red points until germination and with green contours after germination.
    Click OK if asked to switch to Navigation Mode

  2. You now can use the plot windows as navigation panel,

  3. Observe results via ObjectJ>Show ObjectJ Results
  4. Save the project via ObjectJ>Save Project

ObjectJ Menu:

These commands are defined by the embedded macros of SporeTrackerX:

Analyze Spores [F1]

Bright and dark particles will be detected throughout the movie and identified as growing objects. An "object" corresponds to the history of a spore throughout the movie, and is marked with a red "Bright" marker per frame before germination, and with a green "Contour" marker per frame after that as long as it can be traced.
The number label appears in frame#1. Per spore object, the growing area versus time is recorded and stored as string in ObjectJ Results column "AreaPairs". Such a string consists of value pairs separated by semicolon, and each pair is consists of a time and area value separated by a space. This same technique is used for "GrayPairs" to quantify germination (bright to dark transition). After analysis, "Plot_Growth" and "Plot_Germination" are created from the corresponding columns.

Create Plots [F2]

"Plot_Growth" and "Plot_Germination" are created from the corresponding columns. A plot window is a stack holding one plot per object. One additional frame is appended to the stack containing a superimposition of all plots. Typically, a plot window needs not toe be saved, as it can be rebuilt at any time.

Open All Virually [F3]

Opens all linked images as virual stacks, i.e. only a single frame is loaded into RAM. The large stacks should be virutally open so that many operations such as double-clicking a "linked image" or a "linked result" does not loose time loading the entire stack.

Toggle Q flag [Q]

Qualifies/disqualifies the object shown in the current growth plot, sets the plot background to white/ gray, and sets the value in column "Q" to one/zero.

Qualification...

(Dis-)qualifies all objects depending on value in column "Q", and provides the option to kill unqualified objects (which will re-draw the plot windows).

Show Metadata

Shows project path, long and short file names, and batch number. Long file names derived from the label in the stack's metadata.

Set Sign Burst [B]

Use this by activating the Plot_Growth window, position the cursor upon the time where the burst should be marked, and press the shortcut key "B". Any old burst marking will be replaced by the new one, or removed if key was pressed twice on same position.

Set Sign FirstDiv [F]

Similar to "Sign Burst", but intended to mark the first division.

Set Sign T1 [1]

Similar to "Sign Burst", but sets the starting point of the green line whose slope defines doubling time TD.

Set Sign T2 [2]

Similar to "Sign Burst", but sets the end point of the green line whose slope defines doubling time TD.

Activate Navigation [N]

Arranges windows "Plot_Growth" and "Plot_Germination" on the right hand of the screen, and activates the navigation tool in ObjectJ Tools ("N" icon). If an object was selected (e.g. clicked in the ObjectJ results row), the Navigate command activates the plot of this object in both plot stacks. While the "N" tool in ObjectJ Tools is active, the user can click and drag along the time axis of the plot in order to play-back the movie synchronously.