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).
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.
Disclaimer: 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.
- 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.
- In ZEN open the Macro tab (Alt-F8), click on Edit Macro.
Save *.raw data
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.
Troubleshooting the installation
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.
You can try this to fix:
Open a console window (cmd.exe)
Go to the ZEN path:
C:\Windows\Microsoft.NET\Framework\v4.0.30319\RegAsm.exe /tlb /codebase Zeiss.Micro.AIM.ApplicationInterface.dll
- When you uninstall ZEN, make sure to un-register
C:\Windows\Microsoft.NET\Framework\v4.0.30319\RegAsm.exe/u /tlb /codebase Zeiss.Micro.AIM.ApplicationInterface.dll
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 is generated that contains the image coordinates.
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.
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. 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
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.
- FCS positions: These are the scanner positions where the FCS measurements are acquired. They are relative to the current stage position.
- Stage positions: The XY stage and Z focus position.
Stage Z position and FCS Z position are linked. The FCS measurement will always be with respect to the focus and stage position.
BUG When several stage positions are added some positions may end up shifted with respect to the expected location. Therefore, before launching the measurements do visit all stage positions (double click) and verify the correct positioning. Eventually update the position of a wrongly assigned stage position.
- Specify the number and location/type of
FCS points per object
- In the FCS Positions frame the user can
AddFCS points to the current stage position. The other buttons will
Removea highlighted point or
Remove AllFCS points
- In the Stage Positions frame the user can
Addthe current stage (X,Y) and focus (Z) position to the experiment. The user can
Removea highlighted position or
Remove Allpositions. The
Updatebutton will update the highlighted entry to the current position. With a double click the stage moves to the highlighted entry
One image per objectis 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
- Specify output directory, file format, and base file name (an optional prefix to the file name)
- 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
- Total number of FCS measurements for all positions
- If available the user can use DefiniteFocus (hardware autofocus). If the box is checked, then the first Z-stage position will be used as reference for the DefiniteFocus. At every new stage position a focus stabilization step is performed.
FCS Runner generates the following files:
ID_baseFileName.czi|lsm: Image file associated to FCS measurement
ID_baseFileName_postFCS.czi|lsm: Image file associated to FCS measurement. Image has been acquired after the FCS measurement (
image, FCS, imagebutton).
ID_baseFileName_ZENinternalID_R<NrRepetition>_P<ptNr>_K1_Ch<channelNr>.raw: Raw data files containing the FCS data.
ID_baseFileName.xml: Position and type of FCS points with respect to image.
ID_baseFileName.czi|lsm.xml: Stage position associated to image.
ID_baseFileName.txt: Plain txt file containing part of the information in
The ID is an increasing number (0001, 0002,...), the
baseFileName is set in the
File output frame.
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.
fcs file and raw data
The FCSRunner saves the fcs text file and raw data of the measurements (
ID_baseFileName_ZENinternalID_R<NrRepetition>_P<ptNr>_K1_Ch<channelNr>.raw). These files are needed for further processing using programs such as Fluctuation Analyzer.
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.
<?xml version="1.0"?> <xml> <Image Name="C:\MyFolder\0001_baseFileName.czi"></Image> <stage> <x unit="um">0</x> <y unit="um">0</y> <z unit="um">0</z> </stage> <object ID="1"> <class>nuc</class> <x unit="px">255.5</x> <y unit="px">255.5</y> <z unit="px">0</z> </object> ...
Antonio Politi, EMBL, Ellenberg group, updated July 2017