This page describes the elements of the main user interface of TLM-Tracker.
The main window of the program is segmented into several areas, which are numbered from 1-8 on the following figure. The functionality of these areas is described in the following sections. A quick-start guide for the program can be found here.
The task of segmentation and tracking of time-lapse image data is separated by TLM-Tracker into different steps of a workflow. At the beginning, images are loaded using the Menu panel. Images can be inspected and processed using the elements in the General and Toolbar panels.
- Preprocessing: In the first step of the workflow, the images can be cropped, the brightness of all bright field images can be adjusted to one common level, and a range of images can be removed if they are not suitable for the processing.
- Segmentation: The second step of the workflow consists of the segmentation (the recognition) of cells in the different images. There are several pre-defined methods for the segmentation, and it is possible to modify them. In addition, you can create an individual segmentation process adapted to the respective image data. In this step, the properties of the recognized cells like length, width, area and fluorescence intensity are determined.
- Tracking: In the third step, the cells recognized by the segmentation in the second step can be tracked in a time-series of images using different tracking methods. A lineage-tree is created, information on the age of the cell poles is added and the fluorescence intensities can be corrected with regard to the photobleaching effect.
- Export: In the fourth and last step, the achieved data can be exported for further analysis with other programs.
Image data can be loaded as single image files, movie files or as files in the Zeiss AxioVision format ZVI (Zeiss Vision Image) , which is supported using the Bio-Formats Java library .TLM-Tracker supports one bright field and one fluorescence channel. Supported image- and movie-formats depend primarily on the MATLAB version. For MATLAB 7.9 supported file formats are:
- Image files
- BMP, GIF, JPG, PCX, PNG, TIF and others depending on the operating system
- Movie files
- Windows: AVI, MPG, WMV, ASF, ASX
- Linux: AVI, MPG
- Zeiss AxioVision format ZVI
With the "Open images" dialogue you can select image files containing both bright field- and fluorescence image data. If time-lapse image data is loaded by importing the frames from single images, the image files must be loaded in the right chronological order.
When some image files are selected and the dialogue is confirmed, a selection dialogue appears.
Here it is possible to select how the loaded image data is assigned to the bright field and fluorescence channel.
- all bright field images: All images are assigned to the bright field channel.
- all fluorescence images: All images are assigned to the fluorescence channel.
- manual: The images are assigned manually, one by one, to the bright field or fluorescence channel.
- automatic: The images are assigned automatically to the bright field or fluorescence channel. This option works best if there is a significant difference in brightness between the images of different channels.
In case the assignment of images to the referring channel failed it is required to reload these images and assign them, if neccessary, manually to the correct channel.
With this option, projects that were previously created with this program can be loaded. The project file must be located in the same directory as the corresponding image files.
With this option, the settings of a project can be saved at any step of the workflow. The project file must be located in the same directory as the corresponding image files.
Save Screen 1 / Save Screen 2
With this option, the current images of screen 1 and screen 2 can be exported to an image file on the hard disk. Supported export formats are:
- JPG, BMP, PNG, TIF
Export Lineage Tree
With this option, the lineage tree, which can be seen in the tracking-step of the workflow, can be exported as a graph. Supported file formats are:
- GRAPHML, GML
Close all images
This option deletes all present data and resets the program to the state it has when it is started.
This option closes the program. If any data has been loaded a dialogue appears which asks if you want to save the current state of the project before definitely closing the program.
This menu provides the following options:
These options are used to navigate between the different parts of the workflow. Alternatively, you can use the buttons in the Workflow-Panel.
This menu provides the following options:
- Manual: Opens a MATLAB browser window that leads to this manual web page.
- About TLM-Tracker: Shows an info screen that shows the TLM-Tracker version.
Previous / Next
Zoom in / Zoom out
The paint button can be used only in the segmentation mode to correct the image. When this button is pressed, left click on the image will draw a white pixel, right click will draw a black one. This feature can be used to manually separate cells that have been recognized as one cell by the segmentation algorithm.
The fill button can be used only in the segmentation mode. When this button is pressed, left click on the image will fill a black area with white pixels, right click will fill a white area with black pixels. This feature can be used to manually remove objects or cells. This tool will work only on black/white images, not on grayscale images.
The mark background button can be used only in the segmentation mode. If fluorescence image data is present it should be used to mark the background on several positions in the image. These positions should contain image background in all images, so normally it is best to mark the background in the last frame of a time-series. The fluorescence intensity at these positions is used to correct the fluorescence intensity of the cells due to the influence of different expositions.
The edit parameters option is required to adjust the default parameters to the settings of the current time series. The parameters can be changed at any time in workflow process. They are applied by clicking the "OK"-button. If segmentation/tracking data is already present flueorescence adjustment has to be applied using the "Execute"-button.
- time interval in picture series (default 300 sec); only changed if "time interval correction" is activated
- pixel scaling (default 0.1026 micrometer/pixel)
- paint radius for manual pixel painting in segmentation view (default 2 pixels)
If the "Incorporate removed images" box is activated time gaps are calculated for the removed pictures of the time series.
If fluorescence image data is loaded, additional parameters are required to correct the fluorescence intensity values of the cells from the photobleaching effect:
- exposure time [ms]
- basic fluorescence [pixelvalue]: the fluorescence of the cells that can't be bleached by long exposition
- rate constant [s-1]: the kinetic rate constant of the photobleaching effect
The "fluorescence correction" checkbox activates the automatic fluorescence correction in the segmentation/tracking process. The "Execute"-button has to be used to apply changes to already existent segmentation/tracking data.
Details on the impact of the basic fluorescence, the rate constant and the correction of the photobleaching effect can be found here. Both the rate constant and the basic fluorescence can be determined by an experiment with a long exposition time.
The type of images displayed on the two screens differ between different steps of the analysis workflow. The mode of presentation is shown in the title of the screen.
- Preprocessing: Screen 1 and screen 2 show the loaded bright field and fluorescence image data.
- Segmentation: Screen 1 shows the current step in the image processing chain. Screen 2 can be switched between two presentations of the recognized cells in the Display-Panel. In 'Object boxes', the recognized cells are marked with frames on the original image, in 'Object colormap' the recognized cells are shown as colored areas. Under each screen there is a check box named 'Overlap'. If this box is checked, the image on the corresponding screen is overlapped with the previous image. This feature can be used to manually separate cells in the current image that have already been separated in the previous image.
- Tracking: Screen 1 can be switched between two presentations in the Display-Panel. The 'Tracking' mode shows the cells of the current and the last image marked by (blue and green) frames and the assignment of corresponding cells within the two images by (yellow) lines. The 'Cell ages' mode shows the cells of the current image with marked cell poles and ages of the cell poles in cell divisions. Screen 2 shows the complete lineage tree of the cells derived from the analysis of all images, whereas every box marks one cell in one image.
- Export: Screen 1 shows the recognized objects, marked with frames on the original image. Screen 2 can be switched between two presentations in the Display-Panel. The 'Growth' mode shows the growth curves of single cells. The 'Product formation' mode shows the progression of fluorescence intensity for single cells.
The slider in this panel can be used to navigate through the images loaded. Alternatively, an image can be accessed by entering the image index into the edit box. The time point of the current image in the picture series is given in an edit box. It and can be changed at any time.
The elements in this panel can be used to switch between different presentation modes of the screens. The display options depend on the current step in the workflow and are described here.
Workflow step specific panel(s)
The elements in this panel depend on the currently selected workflow step.
- for the Preprocessing step the panel is described here
- for the Segmentation step the panel is described here
- for the Tracking step the panel is described here
- for the Analysis/Export data step the panel is described here
The buttons in this panel can be used to navigate through the different steps of the workflow.