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# <img src = './images/fcsrunner_icon.png'> FcsRunner
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VBA tool to perform imaging and fluorescence correlation spectroscopy measurements (FCS) and automatically save FCS positions to xml file. To be used with Zeiss LSM microscopes running with ZEN black.
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For each stage position user can specify different FCS points. Each position will be visited and imaging and FCS executed. Only one imaging and FCS settings can be used at once.
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<a name=back></a>
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- [Installation and start](#installation)
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- [Running an experiment](#experiment)
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- [File output](#fileoutput)
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> **Disclaimer:**
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> FcsRunner has been tested on Zeiss LSM 780 microscopes with ZEN 2010, 2011, and 2012, and LSM880 microscopes with ZEN2.1 and ZEN2.3. We don’t guarantee that it will work on other configurations and we don’t take any responsibility for damage occuring during or after use of FcsRunner.
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## [<img src="./images/up.PNG">](#back)<a name="installation"></a> Installation and start
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* Copy the file *fcs_runner.lvb* (for ZEN2010 use the file *fcs\_runner\_ZEN2010.lvb* instead) and the directory *resources* to a directory that can be accessed from the computer running ZEN. ./images
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* In ZEN open the *Macro* tab (*Alt-F8*), click on *Edit Macro*.
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Load the macro into ZEN | |
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--------------|----------------------------
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<img src="./images/loadMacro.PNG" width = "400px"> | 1. Load the macro. Browse to the file *fcs\_runner.lvb* <br /> or *fcs\_runner\_ZEN2010.lvb* for older ZEN versions. <br /> 2. Run the macro. <br /> 3. To insert the macro in your list of macros click on the tab.
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<img src="./images/assignMacro.PNG" width = "400px"> | 4. provide a *Menu Entry*, a text, and select *fcs\_runner.lvb* as project.
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### Trouble shooting the installation
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For any issues please contact one of the developers in the Ellenberg group at EMBL, Heidelberg. For errors that appear in the ErrorLog window please provide the PipelineConstructor.err file and the full version of your ZEN software (Help->About). If you have a ZEN version higher than 2010 and the macro complains that it does not find ```Zeiss.Micro.AIM.ApplicationInterface.dll```, you may need to register it manually.
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You can try this to fix
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• Open a console window (cmd.exe)
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• Go to the ZEN path: ``cd ..\..\ZEN``
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• Register the ```Zeiss.Micro.AIM.ApplicationInterface.dll```
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``` C:\Windows\Microsoft.NET\Framework\v4.0.30319\RegAsm.exe /tlb /codebase Zeiss.Micro.AIM.ApplicationInterface.dll ```
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• When you uninstall ZEN, make sure to un-register
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```C:\Windows\Microsoft.NET\Framework\v4.0.30319\RegAsm.exe/u /tlb /codebase Zeiss.Micro.AIM.ApplicationInterface.dll```
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<a name="sectionTwo"></a>
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## [<img src="./images/up.PNG">](#back) <a name=experiment></a>Running an experiment
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Before running an experiment the user first defines imaging and FCS settings in ZEN. The macro will use these settings. When pressing one of the running options (6) the macro will acquire an image and perform the FCS measurements. In additon, an xml file is generated that contains the image coordinates.
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> **Imaging Settings** For FCS calibrated imaging use an uneven number of Z-slices (1, 3, 5...). This ensures that each FCS measurement corresponds to a pixel in the image.
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The user also needs to define to which location each measurement point belongs. Two locations are pre-defined and named `nuc - nucleus` and `cyt - cytoplasm`. The locations are saved as identifier in the [xml file](#xmlfile). The locations can be different than nucleus and cytoplasm depending on the type of sample and the user can use a different name. Note that all software based on data coming from FCSrunner assumes that the locations are named `nuc` and `cyt`.
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The macro allows to acquire FCS measurements at different stage positions. For each stage position the microscope will acquire an image and perform the FCS measurements. The total number of FCS measurements per position must be a multiplier of the total number of FCS measurements per object.
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* **FCS positions**: These are the scanner positions where the FCS measurments are acquired. They are relative to the current stage position.
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* **Stage positions**: The XY stage and Z-focus position.
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FCSRunner commands | |
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:---: | :---
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<img src="./images/fcsRunner.PNG" width = "400px"> | 1. Specify the number and location/type of `FCS points per object` . This information will be written in the [xml file](#xmlfile). <br/> 2. In the **FCS Positions** frame the user can `Add` FCS points to the current stage position. The other buttons will `Remove` an highlighted point or `Remove All` FCS points. In order to simplify the mapping of image and FCS position the macro forces the FCS positions to be in the center of an image pixel. <br/> 3. In the **Stage Positions** frame the user can `Add` the current stage (X,Y) and focus wheel (Z) position to the experiment. The user can `Remove` an highlighted position or `Remove All` positions. The `Update` button will update the highlighted position to the current stage (X,Y) and focus wheel (Z) position. With a double click the microscope move to the highlighted position. <br/> 4. If `One image per object` is checked then the number per FCS points at the each position does not exceed the total number of points per object. <br/> 5. Specify output directory, file format, and base file name (an optional prefix to the file name). <br/> 6. The 3 top buttons start a measurement the last stops a measurement. The difference between buttons is the order in which FCS and imaging are executed <br/> 7. Total number of FCS measurements for all positions
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<a name="sectionThree"></a>
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## [<img src="./images/up.PNG">](#back) <a name="fileoutput"></a> File output
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FCS Runner generates following files
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* `ID_baseFileName.czi|lsm`: Image file associated to FCS measurement (before or after measurement)
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* `ID_baseFileName_postFCS.czi|lsm`: Additional image file acquired after FCS measurement in case button `Image, FCS, image` has been pressed.
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* `ID_baseFileName.fcs` and `ID_baseFileName_ZENinternalID_R<NrRepetition>_P<ptNr>_K1_Ch<channelNr>.raw`: Raw data files containing the FCS data.
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* `ID_baseFileName.xml`: File contains position and type of FCS points with respect to image.
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* `ID_baseFileName.txt`: Plain txt file containing part of the information in `ID_baseFileName.xml`
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The ID is an increasing number (0001, 0002,...)
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### Image file
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As default FCSRunner saves image in *czi* format (`ID_baseFileName.czi|lsm`). The metadata of the image contains the stage and focus position. To get the XYZ coordinates in ZEN load the image and click reuse and then the position checkbox.
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> The position stored in the metadata uses the coordinates as in the ZEN software. For instance the X and Y axis may be mirrored or exchanged.
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### *fcs* file and raw data
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The FCSRunner saves the *fcs* text file and raw data if the measurements (`ID_baseFileName.fcs` and `ID_baseFileName_ZENinternalID_R<NrRepetition>_P<ptNr>_K1_Ch<channelNr>.raw`). This files are needed for further processing using programs such as [Fluctuation Analyzer](https://www.embl.de/~wachsmut/downloads.html).
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### <a name=xmlfile></a> xml file
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Each measurement generates a *xml* file (`ID_baseFileName.xml`) that contains the path to the image associated with the measurement, the stage and focus wheel position. Furthermore, the xml data stores the pixel position and class/location of the FCS measurements. The object ID is > 1 if more than one cell is acquired per image.
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<?xml version="1.0"?>
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<xml>
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<Image Name="C:\MyFolder\0001_baseFileName.czi"></Image>
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<stage>
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<x unit="um">0</x>
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<y unit="um">0</y>
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<z unit="um">0</z>
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</stage>
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<object ID="1">
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<class>nuc</class>
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<x unit="px">255.5</x>
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<y unit="px">255.5</y>
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<z unit="px">0</z>
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</object>
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...
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# Author
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Antonio Politi, EMBL, Ellenberg group, updated June 2017 |
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\ No newline at end of file |