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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 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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VBA tool to perform imaging and fluorescence (cross)correlation spectroscopy (F(C)CS) measurements and automatically save FCS positions to a *xml* file for later reuse. The tool can be used to create data for FCS-calibrated imaging. It 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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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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... | ... | @@ -14,7 +14,7 @@ FCS measurements are performed at multiple stage positions. Typically one stage |
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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 occurring during or after use of FcsRunner.
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> FcsRunner has been tested on Zeiss LSM 780 microscopes with ZEN 2010, 2011 and 2012 as well as 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 occurring during or after the use of FcsRunner.
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... | ... | @@ -34,8 +34,8 @@ FCS measurements are performed at multiple stage positions. Typically one stage |
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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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For any issues please contact one of the developers in the Ellenberg group at EMBL, Heidelberg and provide the version of the software 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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### Trouble-shooting the installation
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For any issues please contact one of the developers in the Ellenberg group at EMBL, Heidelberg; and provide the version of the software 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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... | ... | @@ -54,29 +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, a [xml file](#xmlfile) 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) 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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* **FCS positions**: These are the scanner positions where the FCS measurements 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 new 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`. Then add new FCS positions.
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<p align = 'center'><img src="./images/fcsRunner.PNG" width = "400px"></p>
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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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2. In the **FCS Positions** frame the user can `Add` FCS points to the current stage position. The other buttons will `Remove` a 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` a 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, the number of FCS points at each position does not exceed the total number of points per object. A new stage position 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 button 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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... | ... | @@ -84,7 +84,7 @@ The macro allows to acquire FCS measurements at different stage positions. For e |
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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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FCS Runner generates the following files:
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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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... | ... | @@ -96,7 +96,7 @@ FCS Runner generates following files |
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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`). The metadata of the image `ID_baseFileName.czi.xml` contains the stage and focus position.
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As default FCSRunner saves images in *czi* format (`ID_baseFileName.czi`). The metadata of an 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 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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