... | ... | @@ -9,10 +9,10 @@ The repository [adaptive\_feedback\_mic\_fiji](https://git.embl.de/politi/adapti |
|
|
|
|
|
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
|
|
|
1. Gaussian blur and threshold using a specified 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
|
|
|
4. If an object is found send stage position(s) and/or FCS position(s) (computed with respect to the object of interest) to MyPiC
|
|
|
5. Send to MyPiC which action to perform, e.g. update stage position for tracking or FCS measurements
|
|
|
|
|
|
|
... | ... | @@ -27,7 +27,7 @@ Install [FiJi](http://fiji.sc/#download) to a directory where you have writing r |
|
|
## Compilation of the jar files
|
|
|
1. Install [maven](https://maven.apache.org/index.html)
|
|
|
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.
|
|
|
3. Change directory to `automatedfcs` and type `mvn package` to compile the package. If the compilation is successful the jar file is stored in a newly created `automatedfcs\target` directory.
|
|
|
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](https://commons.apache.org/proper/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.
|
... | ... | @@ -40,7 +40,7 @@ The wiki contains following sections: |
|
|
|
|
|
* [**Start the plugin**](#start)
|
|
|
* [**Directory to monitor and parameters GUI**](#dirmon)
|
|
|
* [**Image to analyse parameters**](#image)
|
|
|
* [**Image to analyze parameters**](#image)
|
|
|
* [**Segmentation parameters**](#segmentation)
|
|
|
* [**FCS measurement points parameters**](#fcspos)
|
|
|
* [**Test and save settings commands**](#save)
|
... | ... | @@ -50,6 +50,7 @@ The wiki contains following sections: |
|
|
You can start the plugin from `Plugins > EMBL > Automated Fcs`.
|
|
|
|
|
|
## [<img src="./images/up.PNG">](#back) <a name="dirmon"></a> Directory to monitor and parameters GUI
|
|
|
|
|
|
This directory is the directory where MyPiC saves the data.
|
|
|
|
|
|
Main window | Parameter window|
|
... | ... | @@ -61,13 +62,13 @@ Main window | Parameter window| |
|
|
2. Click on `Parameters`
|
|
|
3. This will open a window where the user can enter the specifications for the image analysis
|
|
|
|
|
|
## [<img src="./images/up.PNG">](#back) <a name = "image"></a>Image to analyse parameters
|
|
|
## [<img src="./images/up.PNG">](#back) <a name = "image"></a>Image to analyze parameters
|
|
|
|
|
|
|
|
|
<div align = "center" > <img src = './images/AutomatedFCS_GUI_Artboard3.png' width = "400px" > </div>
|
|
|
|
|
|
|
|
|
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`. Commands and parameters are written in the WindowsRegistry
|
|
|
The user can specify up to 3 images that will be analyzed by the plugin (Job1-3). The images should match to MyPiC task where processing is set to `Online image Analysis`. Commands and parameters are written in the WindowsRegistry
|
|
|
|
|
|
```
|
|
|
HKEY_CURRENT_USER\Software\VB and VBA Program Settings\OnlineImageAnalysis\macro
|
... | ... | @@ -75,16 +76,16 @@ HKEY_CURRENT_USER\Software\VB and VBA Program Settings\OnlineImageAnalysis\macro |
|
|
|
|
|
for MyPiC to read.
|
|
|
|
|
|
1. **Pipeline** Name of MyPiC pipeline to analyse
|
|
|
1. **Pipeline** Name of MyPiC pipeline to analyze
|
|
|
* **None**: do not perform any analysis
|
|
|
* **Default**: Analyse files containing string `*DE*` from default pipeline
|
|
|
* **Trigger1**: Analyse files containing name `*TR1*` from Trigger1 pipeline
|
|
|
* **Trigger2**: Analyse files containing name `*TR2*` from Trigger2 pipeline
|
|
|
* **Default**: Analyze files containing string `*DE*` from default pipeline
|
|
|
* **Trigger1**: Analyze files containing name `*TR1*` from Trigger1 pipeline
|
|
|
* **Trigger2**: Analyze files containing name `*TR2*` from Trigger2 pipeline
|
|
|
|
|
|
2. **Task**: This is the image number in the order of acquisition of the pipeline
|
|
|
3. **Command**: Command passed to MyPiC upon succesful segmentation. Written in the windows registry **CodeMic**.
|
|
|
3. **Command**: Command passed to MyPiC upon successful segmentation. Written in the windows registry **CodeMic**.
|
|
|
* `nothing`: do not perform any action
|
|
|
* `focus`: Useful for object based tracking in 3D. Compute center of mass of segmented binary object and pass coordinats to MyPiC (windows registry **X**, **Y**, **Z**). Microscopy position are updated if MyPiC options **TrackXY** and/or **TrackZ** are on .
|
|
|
* `focus`: Useful for object based tracking in 3D. Compute center of mass of segmented binary object and pass coordinates to MyPiC (windows registry **X**, **Y**, **Z**). Microscopy position are updated if MyPiC options **TrackXY** and/or **TrackZ** are on .
|
|
|
* `setFcsPos`: Pass coordinates for FCS measurements to MyPiC (registry **fcsX**,**fcsY**,**fcsZ**). Number of points and positions are specified in [FCS measurement points parameters](#fcspos). MyPiC starts FCS measurements at these positions if the next task is a FCS job
|
|
|
* `setFcsPos;focus`: Combination of 2 commands. MyPiC first performs FCS measurements and then updates the stage position according to the center of mass of segmented object
|
|
|
* `trigger1`: MyPiC starts Trigger1 pipeline at XYZ of center of mass of segmented object(s). Several stage positions can be specified at once using **Number of particles** >1
|
... | ... | @@ -94,7 +95,7 @@ for MyPiC to read. |
|
|
|
|
|
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.
|
|
|
|
|
|
In object detection allows for object based tracking of a single cell using a nearest neighbour algorithm (Command: **focus**). This can be used to perform long term high-resolution imaging of objects that are moving in space using a small field of view.
|
|
|
In object detection allows for object based tracking of a single cell using a nearest neighbor algorithm (Command: **focus**). This can be used to perform long term high-resolution imaging of objects that are moving in space using a small field of view.
|
|
|
|
|
|
<div align = "center" > <img src = './images/AutomatedFCS_GUI_Artboard4.png' width = "400px" > </div>
|
|
|
|
... | ... | @@ -102,7 +103,7 @@ In object detection allows for object based tracking of a single cell using a ne |
|
|
|
|
|
1. **Channel segmentation**: Channel to use for object segmentation
|
|
|
2. **Seg. Method**: Method to find threshold to separate foreground and background pixels. The name refers to the method as implemented in ImageJ
|
|
|
3. **filter radius (px)** Radius in pixels of the gaussian and median filters applied on the image before thresholding
|
|
|
3. **filter radius (px)** Radius in pixels of the Gaussian and median filters 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
|
... | ... | @@ -128,7 +129,7 @@ Note that FCS measurements are only performed by the microscope if the plugin se |
|
|
2. **# oper. (erode < 0, dilate > 0)**: Number of pixels to erode or dilate depending on the sign. With negative values the FCS points are placed withing the object of interest
|
|
|
3. **FCS pts. region 2 (outside)**: umber of FCS in a region around the object of interest. The id of these points is *cyt* (cytoplasm)
|
|
|
4. **# oper. (erode < 0, dilate > 0)**: Number of pixels to erode or dilate depending on the sign. With positive values the FCS points are placed outside the 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
|
|
|
5. **Update z-pos for FCS**: If *Yes* FCS measurements are performed on a new Z-position set from the center of mass of the segmented object of interest
|
|
|
|
|
|
## [<img src="./images/up.PNG">](#back) <a name=save></a> Test and save settings commands
|
|
|
|
... | ... | @@ -146,14 +147,14 @@ To start the plugin and automatically process files created in the directory to |
|
|
|
|
|
To process a whole directory and test the segmentation pipeline press **RunOnDir**.
|
|
|
|
|
|
Segmentation jpg are saved in the directories specified in **Analyze results saving**
|
|
|
When an objects of interest are found segmentation results jpg files are saved in the directories specified in **Analyze results saving**
|
|
|
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
|
|
## [<img src="./images/up.PNG">](#back) <a name=start></a>Example output for FCS-calibrated imaging
|
|
|
We show an example for Automated FCS using these parameters
|
|
|
We show an example for Automated FCS using these parameters
|
|
|
|
|
|
<img src = './images/AutomatedFCS_GUI_Artboard2.png' width = "400px" >
|
|
|
|
... | ... | @@ -167,13 +168,13 @@ Cells express a fluorescent protein and their DNA has been stained with SiR-DNA. |
|
|
* task2: A high resolution imaging of the fluorescent protein (Channel 1) and DNA (Channel 3)
|
|
|
* task3: FCS measurements
|
|
|
|
|
|
The Automated FCS performs:
|
|
|
* Job1: detects cells using the DNA stain. Choose cells where the fluorescent protein is within a certain range. The coordinates of cells that fulfill all criteria are passed to MyPiC to start the Trigger1 pipeline.
|
|
|
The *Automated FCS* plugins performs:
|
|
|
* Job1: Cell detection using DNA channel. Cells where the fluorescent protein is within a certain range are selected. The coordinates of cells that fulfill all criteria are passed to MyPiC to start the Trigger1 pipeline.
|
|
|
|
|
|
Output generated for every processed image | Output generated when objects of interest are found
|
|
|
:--- | :---
|
|
|
<img src = './images/example_output.png' width = "400px" > | <img src = './images/example_output2.png' width = "400px" >
|
|
|
Segmenation of the DNA channel (red) returned 16 ROIs. <br/>Only for ROI 10 and 13 is the intensity of the fluorescent protein (green) within the specified range. | The ROI for the two cells picked are shown in green. <br/> The coordinates are passed to MyPiC to execute the Trigge1 pipleline for each position
|
|
|
Segmentation of the DNA channel (red) returned 16 ROIs. <br/>Only for ROI 10 and 13 is the intensity of the fluorescent protein (green) within the specified range. | The ROI for the two cells picked are shown in green. <br/> The coordinates are passed to MyPiC to execute the Trigge1 pipeline for each position
|
|
|
|
|
|
* Job2: detects cells using the DNA stain. Chooses the cell closest to the center where the fluorescent protein is within a certain range. Determines the coordinates of the FCS measurements and pass their values to MyPiC. In task 3 of Trigger1 MyPiC will start FCS measurements at these positions.
|
|
|
|
... | ... | |