Running the Automated FCS plugin

Start the plugin

You can start the plugin from Plugins > EMBL > Automated Fcs.

Directory to monitor and parameters

This directory is the directory where MyPiC stores the data.

1. In the field Directory to monitor specify the directory where the data from MyPiC is stored
2. Click on Parameters
3. This will open a window where the user can enter the specifications for the image analysis

Image to analyse

The user can specify up to 3 images that will be analysed by the plugin (Job1-3). The images should match to MyPiC task where process is set to Online image Analysis

1. Image Name of pipeline from MyPiC to analyse
   None - do not perform any analysis
   Default - Files with name *DE* from default pipeline
   Trigger1 (Trigger2) - Files with name *TR1* (*TR2*) from Trigger1 (Trigger2) pipeline
2. Img. Task Task to be analysed. This is the image id in the order of acquisition. For example files with name DE_2_*
3. Command Command to pass to MyPiC
   nothing - do not perform any action
   focus - compute XYZ of center of mass of the segmented binary object. MyPiC updates the stage position accordingly. This is used for tracking objects in 3D.
   setFcsPos - pass XYZ position(s) for FCS measurements specified in Location of FCS. MyPiC starts FCS measurements at those positions
   setFcsPos;focus - MyPiC first performs FCS measurements and then updates the stage position
   trigger1 (trigger2) - MyPiC starts Trigger1 (Trigger2) pipeline at XYZ of center of mass of segmented object(s). Several stage positions can be specified.

Specify segmentation paremeters

1. Main seg. Channel Channel where to perform segmentation.
2. Seg. Method Method to find threshold to separate foreground and background pixels. The name refer to the method as implemented in imageJ.
3. filter radius (px) Radius in pixels of the gaussian filter applied on the image before thresholding.
4. Exclude objects > (um2) Exclude objects above this area-size in micrometer^2
5. Exclude objects < (um2) xclude objects below this area-size in micrometer^2
6. Watershed if > (um2) Perform a watershed operation on objects that exceed this are. This parameter is useful to separate objects that are in close proximity.
7. Min mean intensity The mean fluorescence intensity for each object of interest must be above this value.
8. Max mean intensity The mean fluorescence intensity for each object of interest must be below this value.
9. Exclude boundaries If yes object that touch the boundary are excluded
10. Number of particles Pass coordinates to MyPiC of maximally this number of objects
11. Channels intensity filter1/2 Specify one or two additional channels for which a range in mean fluorescence intensity should be fulfilled.
12. Pick particle in
    center choose one particle that fulfills all the limits in area and fluorescene intensity closest to the center of the image. This allows for tracking the same object through time and space.
    random choose one particle that fulfills all the limits in area and fluorescene intensity at random.
    many choose several particle that fulfill the limits in area and fluorescene intensity at random. The maximal number of particles is specified by Number of particles.

Location of FCS measurement points

Note that FCS measurements are only performed by MyPiC if the Command setFcsPos or setFcsPos;focus is specified. The points are placed so that they are as distant as possible. For example for 4 points the FCS points are located on 2 orthogonal lines.

1. FCS pts. inside obj Number of FCS points inside object of interest. The id of these points is nuc for nucleus
2. # pixel erode Number of erosion operations of segmented object of interest
3. FCS pts. outside obj Number of FCS points inside object of interest. The id of these points is cyt for cytoplasm
4. # pixel dilate Number of dilation operations of segmented object of interest.
5. Update z-pos for FCS If Yes the the FCS measurments are performed on a new Z-position estimated from the center of mass of the segmented object of interest.

Test and save settings

1. Run on file Test the analysis operations on a user defined file. The program will prompt for a file.
2. Load Para Load parameter from a file named Automated_FCS.ini. Such a file is automatically generated when the monitoring of the directory is started.
3. Save Para Save parameters to a manually specified *.ini file
4. OK Close window and go back to main menu

Start monitoring

To start the plugin and automatically process files created in the directory to monitor press Start.

If analyze only new files is clicked only new generated files are processed. In case you restart MyPiC in the same folder the old files need to be deleted. If this option is not on, the plugin looks for changes in the files. In this situation it can occur that the plugin processes one file twice.