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Last edited by Antonio Politi

Adaptive Feedback Microscopy ImageJ/FiJi utilities

The repository adaptive_feedback_mic_fiji contains ImageJ/FiJi tools to be used with the Zeiss VBA macro MyPiC to perform adaptive feedback microscopy experiments.

Adaptive feedback microscopy Experiments where the microscopy software interacts with an image analysis software. The image is processed and depending on the results a different imaging protocol is started.

The Automated FCS plugin monitors a folder for new images generated by MyPiC and the LSM microscope. When a file matches a specified task in a pipeline the image is processed according to:

  1. Gaussian blurr and threhsold using a specifed channel of the image
  2. Seeded watershed for objects that exceed a certain area size
  3. Pick objects of interest that are within an area size range and fluorescence intensity range
  4. If an object is found send stage position(s) and/or FCS position(s) (computed with respect to the object of interset) to MyPiC
  5. Send to MyPiC which action to perform, e.g. update stage position for tracking or FCS measurements

Installation

Install FiJi to a directory where you have writing rights.

Without compiling the jar files

  1. Create a directory in the FiJi plugin directory e.g. Fiji.app\plugins\EMBL
  2. Copy the jar files and the python file contained in the dist directory of this repository to the newly create directory.

Compilation of the jar files

  1. Install maven
  2. Open a command line tool.
  3. Change directory to automatedfcs and type mvn package to compile the package. If the compilation is succesful the jar file is stored in a newly created automatedfcs\target directoy.
  4. Repeat point 3. in the directory segment_Particles and CommMicroscope.
  5. Copy the jar files and the python file located in the dist directory to the FiJi plugins directory as explained before.
  6. Get the commons-io java library (version >= 2.5, the plugin has been tested with common-io-2.5.jar) and copy the jar file to the FiJi plugins directory.

Running the Automated FCS plugin

The wiki contains following sections:

Start the plugin

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

Directory to monitor and parameters GUI

This directory is the directory where MyPiC saves the data.

Main window Parameter window
  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 parameters

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 processing 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 number in the order of acquisition of the pipeline
  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 FCS measurement points. MyPiC starts FCS measurements at these positions
    setFcsPos;focus - MyPiC first performs FCS measurements and then updates the stage position according to the center of mass of segmented object
    trigger1 (trigger2) - MyPiC starts Trigger1 (Trigger2) pipeline at XYZ of center of mass of segmented object(s). Several stage positions can be specified at once using Number of particles >1 and Pick particle in = many

Segmentation parameters

After segmentation objects that are within a certain area range and fluorescence intensity range are selected. From these objects the plugin picks one or more objects to be used for specifying imaging coordinates and FCS positions.

  1. Main seg. Channel Channel to use for object 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 area-size in micrometer^2
  5. Exclude objects < (um2) Exclude objects below area-size in micrometer^2
  6. Watershed if > (um2) Perform a watershed operation on objects that exceed area. 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 objects touching the boundary are excluded
  10. Number of particles Maximally allow for this number of objects
  11. Channels intensity filter1/2 Specify one or two additional channels for which the fluorescence intensity must be in a certain range
  12. Pick particle in
    center choose one particle closest to the center of the image. This allows for tracking an object through time and space.
    random choose one particle at random.
    many choose several particle at random. The maximal number of particles is specified by Number of particles

The default Max mean intensity values are for 8bit images. This needs to be increased for 12bit (maximum intensity 4093) or 16bit (maximum intensity 65536) images

FCS measurement points parameters

Note that FCS measurements are only performed by the microscope if the plugin sends the command setFcsPos or setFcsPos;focus to MyPiC. 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. The user can specify the distance from the boundary of the segmented primary object.

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

Test and save settings commands

  1. Run on file Test the analysis pipeline on a user defined file. The program will prompt for a file
  2. Load Para Load parameters from a file named Automated_FCS.ini. Such a file is automatically generated after pressing Start.
  3. Save Para Save parameters to a manually specified *.ini file
  4. OK Close window and go back to main window

Start monitoring commands

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 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.