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# <img src = './images/fcsrunner_icon.png'> FcsRunner
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<a name=back></a>
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VBA tool to perform imaging and fluorescence (cross)correlation spectroscopy measurements (F(C)CS) and automatically save FCS positions to xml file. To be used with Zeiss LSM microscopes running with ZEN black (version >= 2010). The tool can be used to create data for FCS-calibrated imaging.
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VBA tool to perform imaging and fluorescence (cross)correlation spectroscopy measurements (F(C)CS) and automatically save FCS positions to a *xml* file for later reuse. The tool can be used to create data for FCS-calibrated imaging. Requires Zeiss LSM microscopes running with ZEN black (version >= 2010).
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FCS measurements are performed at multiple stage positions. Typically one stage position per cell. At each stage position the user specifies FCS points. After all positions have been specified the users starts the acquisition. Then for each position an image is acquired and FCS measurements are performed. The image and FCS settings are given by the current settings in ZEN.
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... | ... | @@ -25,14 +24,14 @@ FCS measurements are performed at multiple stage positions. Typically one stage |
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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.
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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 = "800px"> | 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 = "800px"> | 4. Provide a *Menu Entry*, a text, and select *fcs\_runner.lvb* as project.
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|Load the macro into ZEN | Explanations of the buttons |
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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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### Save *.raw data
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The compute bleach corrected correlation functions from the photon counts some programs require the raw data. In ZEN you need to specify this in the *Maintain* menu. A directory does not need to be specified. FCSRunner copies the **.raw* files in the current directory.
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To compute bleach corrected correlation functions some programs require the photon counts raw data. In ZEN you need to specify this in the *Maintain* menu. A directory does not need to be specified. FCSRunner copies the **.raw* files in the current directory.
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<p align = 'center'> <img src="./images/rawcounts.PNG" width = "300px"> </p>
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### Trouble shooting the installation
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... | ... | @@ -55,22 +54,29 @@ You can try this to fix |
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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 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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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 the macro will acquire an image and perform the FCS measurements. In additon, a [xml file](#xmlfile) 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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> **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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> Do not move the stage or focus position when placing FCS points for a cell. If you need to correct an imaging position: remove all FCS positions, change stage and focus positions and press `Update`, add the FCS positions
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> Do not move the stage or focus position when placing FCS points for a cell. If you need to correct an imaging position: remove all FCS positions, change stage and focus positions and press `Update`, add new FCS positions.
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<p align = 'center'><img src="./images/fcsRunner.PNG" width = "400px"></p>
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FCSRunner commands | Explanations
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:-----------------------------------------:|:---------
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<img src="./images/fcsRunner.PNG" width = "2000px"> | 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. 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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1. Specify the number and location/type of `FCS points per object`.
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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.
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3. In the **Stage Positions** frame the user can `Add` the current stage (X,Y) and focus (Z) position to the experiment. The user can `Remove` an highlighted position or `Remove All` positions. The `Update` button will update the highlighted entry to the current position. With a double click the stage moves to the highlighted entry. <br/>
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4. If `One image per object` is checked then the number of FCS points at each position does not exceed the total number of points per object. A new stage positions entry is automatically generated.
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5. Specify output directory, file format, and base file name (an optional prefix to the file name).
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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.
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7. Total number of FCS measurements for all positions
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... | ... | @@ -80,24 +86,23 @@ The macro allows to acquire FCS measurements at different stage positions. For e |
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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.czi|lsm`: Image file associated to FCS measurement
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* `ID_baseFileName_postFCS.czi|lsm`: Image file associated to FCS measurement. Image has been acquired after the FCS measurement (`FCS, image` and `image, FCS, image` button).
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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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* `ID_baseFileName.xml`: Position and type of FCS points with respect to image.
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* `ID_baseFileName.czi|lsm.xml`: Stage position associated 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,...), the `baseFileName` is set in the `File output` frame
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The ID is an increasing number (0001, 0002,...), the `baseFileName` is set in the `File output` frame.
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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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As default FCSRunner saves image in *czi* format (`ID_baseFileName.czi`). The metadata of the image `ID_baseFileName.czi.xml` contains the stage and focus position.
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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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The FCSRunner saves the *fcs* text file and raw data of the measurements (`ID_baseFileName.fcs` and `ID_baseFileName_ZENinternalID_R<NrRepetition>_P<ptNr>_K1_Ch<channelNr>.raw`). These 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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### <a name=xmlfile></a> *xml* file associated with FCS measurement
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Each FCS 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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