|
|
# Running the Automated FCS plugin
|
|
|
## Start the plugin
|
|
|
Adaptive Feedback Microscopy ImageJ/FiJi utilities
|
|
|
========
|
|
|
|
|
|
The repository [adaptive\_feedback\_mic\_fiji](https://git.embl.de/politi/adaptive_feedback_mic_fiji) contains ImageJ/FiJi tools to be used with the Zeiss VBA macro [MyPiC](https://git.embl.de/politi/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
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
# [<img src="./images/up.PNG">](#back) <a name="installation"></a> Installation
|
|
|
Install [FiJi](http://fiji.sc/#download) 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](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.
|
|
|
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.
|
|
|
|
|
|
# Running the Automated FCS plugin
|
|
|
|
|
|
The wiki contains following sections:
|
|
|
|
|
|
<a name="back"></a>
|
|
|
|
|
|
* [**Start the plugin**](#start)
|
|
|
* [**Directory to monitor and parameters GUI**](#dirmon)
|
|
|
* [**Image to analyse parameters**](#image)
|
|
|
* [**Segmentation parameters**](#segmentation)
|
|
|
* [**FCS measurement points parameters**](#fcspos)
|
|
|
* [**Test and save settings commands**](#save)
|
|
|
* [**Start monitoring commands**](#start)
|
|
|
|
|
|
## [<img src="./images/up.PNG">](#back) <a name="start"></a>Start the plugin
|
|
|
You can start the plugin from `Plugins > EMBL > Automated Fcs`.
|
|
|
|
|
|
## Directory to monitor and parameters
|
|
|
## [<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|
|
|
|
Main window | Parameter window|
|
|
|
:---: | :---:
|
|
|
<img src = './images/automatedfcs1.png' width = "400px" > | <img src = './images/automatedfcs2.png' width = "400px" >
|
|
|
<img src = './images/AutomatedFCS_GUI_Artboard1.png' width = "400px" > | <img src = './images/AutomatedFCS_GUI_Artboard2.png' width = "400px" >
|
|
|
|
|
|
|
|
|
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
|
|
|
## [<img src="./images/up.PNG">](#back) <a name = "image"></a>Image to analyse parameters
|
|
|
|
|
|
|
|
|
<div align = "center" > <img src = './images/automatedfcs2_1.png' width = "400px" > </div>
|
|
|
<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`
|
... | ... | @@ -26,11 +75,11 @@ The user can specify up to 3 images that will be analysed by the plugin (Job1-3) |
|
|
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 <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*
|
|
|
|
|
|
## Segmentation parameters
|
|
|
## [<img src="./images/up.PNG">](#back) <a name="segmentation"></a> 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.
|
|
|
|
|
|
<div align = "center" > <img src = './images/automatedfcs2_2.png' width = "400px" > </div>
|
|
|
<div align = "center" > <img src = './images/AutomatedFCS_GUI_Artboard4.png' width = "400px" > </div>
|
|
|
|
|
|
|
|
|
|
... | ... | @@ -50,10 +99,10 @@ After segmentation objects that are within a certain area range and fluorescence |
|
|
> 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
|
|
|
>
|
|
|
|
|
|
## <a name=fcspos></a>FCS measurement points
|
|
|
## [<img src="./images/up.PNG">](#back) <a name=fcspos></a> 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.
|
|
|
|
|
|
<div align = "center" > <img src = './images/automatedfcs2_3.png' width = "400px" > </div>
|
|
|
<div align = "center" > <img src = './images/AutomatedFCS_GUI_Artboard5.png' width = "400px" > </div>
|
|
|
|
|
|
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
|
... | ... | @@ -61,17 +110,17 @@ Note that FCS measurements are only performed by the microscope if the plugin se |
|
|
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
|
|
|
## [<img src="./images/up.PNG">](#back) <a name=save></a> Test and save settings commands
|
|
|
|
|
|
<div align = "center" > <img src = './images/automatedfcs2_4.png' width = "400px" > </div>
|
|
|
<div align = "center" > <img src = './images/AutomatedFCS_GUI_Artboard6.png' width = "400px" > </div>
|
|
|
|
|
|
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`](#start).
|
|
|
3. `Save Para` Save parameters to a manually specified **.ini* file
|
|
|
4. `OK` Close window and go back to main window
|
|
|
|
|
|
## <a name=start></a>Start monitoring
|
|
|
<div align = "center" > <img src = './images/automatedfcs3.png' width = "400px" > </div>
|
|
|
## [<img src="./images/up.PNG">](#back) <a name=start></a>Start monitoring commands
|
|
|
<div align = "center" > <img src = './images/AutomatedFCS_GUI_Artboard7.png' width = "400px" > </div>
|
|
|
|
|
|
To start the plugin and automatically process files created in the directory to monitor press `Start`.
|
|
|
|
... | ... | |