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You can start the plugin from `Plugins > EMBL > Automated Fcs`.
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## Directory to monitor and parameters
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This directory is the directory where MyPiC stores the data.
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This directory is the directory where MyPiC saves the data.
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| Main window | Parameter window|
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:---: | :---:
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<img src = './images/automatedfcs1.png' width = "400px" > | <img src = './images/automatedfcs2.png' width = "400px" >
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<img src = './images/automatedfcs1.png' width = "400px" > <img src = './images/automatedfcs2.png' width = "400px" >
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1. In the field `Directory to monitor` specify the directory where the data from MyPiC is stored
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2. Click on `Parameters`
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<div align = "center" > <img src = './images/automatedfcs2_1.png' width = "400px" > </div>
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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`
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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`
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1. `Image` Name of pipeline from MyPiC to analyse <br/>*None* - do not perform any analysis <br/> *Default* - Files with name `*DE*` from default pipeline <br/> *Trigger1* (*Trigger2*) - Files with name `*TR1*` (`*TR2*`) from Trigger1 (Trigger2) pipeline
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2. `Img. Task` Task to be analysed. This is the image id in the order of acquisition. For example files with name `DE_2_*`
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3. `Command` Command to pass to MyPiC <br/> *nothing* - do not perform any action <br/> *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. <br/> *setFcsPos* - pass XYZ position(s) for FCS measurements specified in [Location of FCS](#fcspos). MyPiC starts FCS measurements at those positions <br/> *setFcsPos;focus* - MyPiC first performs FCS measurements and then updates the stage position <br/> *trigger1* (*trigger2*) - MyPiC starts Trigger1 (Trigger2) pipeline at XYZ of center of mass of segmented object(s). Several stage positions can be specified.
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2. `Img. Task` Task to be analysed. This is the image number in the order of acquisition of the pipeline
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3. `Command` Command to pass to MyPiC <br/> *nothing* - do not perform any action <br/> *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. <br/> *setFcsPos* - pass XYZ position(s) for FCS measurements specified in [FCS measurement points](#fcspos). MyPiC starts FCS measurements at these positions <br/> *setFcsPos;focus* - MyPiC first performs FCS measurements and then updates the stage position according to the center of mass of segmented object <br/> *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`](#pickpoints) = *many*
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## Segmentation parameters
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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.
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## Specify segmentation paremeters
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<div align = "center" > <img src = './images/automatedfcs2_2.png' width = "400px" > </div>
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1. `Main seg. Channel` Channel where to perform segmentation.
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2. `Seg. Method` Method to find threshold to separate foreground and background pixels. The name refer to the method as implemented in imageJ.
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3. `filter radius (px)` Radius in pixels of the gaussian filter applied on the image before thresholding.
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4. `Exclude objects > (um2)` Exclude objects above this area-size in micrometer^2
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5. `Exclude objects < (um2)` xclude objects below this area-size in micrometer^2
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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.
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1. `Main seg. Channel` Channel to use for object segmentation
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2. `Seg. Method` Method to find threshold to separate foreground and background pixels. The name refer to the method as implemented in ImageJ.
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3. `filter radius (px)` Radius in pixels of the gaussian filter applied on the image before thresholding
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4. `Exclude objects > (um2)` Exclude objects above area-size in micrometer^2
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5. `Exclude objects < (um2)` Exclude objects below area-size in micrometer^2
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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.
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7. `Min mean intensity` The mean fluorescence intensity for each object of interest must be above this value.
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8. `Max mean intensity` The mean fluorescence intensity for each object of interest must be below this value.
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9. `Exclude boundaries` If yes object that touch the boundary are excluded
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10. `Number of particles` Pass coordinates to MyPiC of maximally this number of objects
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11. `Channels intensity filter1/2` Specify one or two additional channels for which a range in mean fluorescence intensity should be fulfilled.
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12. `Pick particle in` <br/> *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. <br/> *random* choose one particle that fulfills all the limits in area and fluorescene intensity at random. <br/> *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`.
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9. `Exclude boundaries` If yes objects touching the boundary are excluded
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10. `Number of particles` Maximally allow for this number of objects
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11. `Channels intensity filter1/2` Specify one or two additional channels for which the fluorescence intensity must be in a certain range
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12. <a name=pickpoints></a>`Pick particle in` <br/> *center* choose one particle closest to the center of the image. This allows for tracking an object through time and space. <br/> *random* choose one particle at random. <br/> *many* choose several particle at random. The maximal number of particles is specified by `Number of particles`
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## <a name=fcspos></a>Location of FCS measurement points
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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.
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> 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
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>
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## <a name=fcspos></a>FCS measurement points
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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.
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<div align = "center" > <img src = './images/automatedfcs2_3.png' width = "400px" > </div>
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1. `FCS pts. inside obj` Number of FCS points inside object of interest. The id of these points is *nuc* for nucleus
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1. `FCS pts. inside obj` Number of FCS points inside object of interest. The id of these points is *nuc* (nucleus)
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2. `# pixel erode` Number of erosion operations of segmented object of interest
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3. `FCS pts. outside obj` Number of FCS points inside object of interest. The id of these points is *cyt* for cytoplasm
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4. `# pixel dilate` Number of dilation operations of segmented object of interest.
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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.
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3. `FCS pts. outside obj` Number of FCS points inside object of interest. The id of these points is *cyt* (cytoplasm)
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4. `# pixel dilate` Number of dilation operations of the segmented object of interest
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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
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## Test and save settings
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<div align = "center" > <img src = './images/automatedfcs2_4.png' width = "400px" > </div>
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1. `Run on file` Test the analysis operations on a user defined file. The program will prompt for a file.
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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.
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1. `Run on file` Test the analysis pipeline on a user defined file. The program will prompt for a file
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2. `Load Para` Load parameters from a file named *Automated_FCS.ini*. Such a file is automatically generated after pressing [`Start`](#start).
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3. `Save Para` Save parameters to a manually specified **.ini* file
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4. `OK` Close window and go back to main menu
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4. `OK` Close window and go back to main window
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## Start monitoring
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## <a name=start></a>Start monitoring
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<div align = "center" > <img src = './images/automatedfcs3.png' width = "400px" > </div>
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To start the plugin and automatically process files created in the directory to monitor press `Start`.
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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.
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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.
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