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To measure a sample with more than one microscopes within the same project, or if the sample was removed from the stage for some reason, the new coordinates of the particles need to be calculated.
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# Recalculate coordinate system
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# Import an image from Zeiss Zen
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It is possible to import (fluorescence) microscopy projects from Zeiss Zen® to Gepard, e.g. to make a correlative fluorescence/Raman measurement.
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## On the optical microscope
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If you have a sample holder with dedicated markers for correlative spectroscopy, you should perform a calibration of the coordinate system prior to image acquisition.
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Record an EDF (enriched depth of focus) image. In this mode a stack of images at varying focus positions is acquired. The images are then combined into a maximal contrast image. As method choose "Wavelets (with heigtmap)". For details regarding the measurement setup, please refer to the documentation of your microscope.
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By the end of the measurement, your Zen project should contain two channels: the an EDF image and a height map. Latter is a greyscale image, in which focus positions are indicated as different shades of grey.
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Export the project as single channel images. Choose the option to export metadata in an xml file.
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After exporting, four files should have been created: Two tiff files, containing the EDF image and the height map, respectively, and two xml files containing metadata.
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## Import to Gepard
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Launch Gepard and choose `Import Zeiss project`on the left menu bar. In the pop-up file dialog, choose the metadata file ending in "metadata.xml".
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Gepard loads the two images and reads relevant metadata (z value range of the height map, coordinates of the markers) from the xml file. After that, you will be prompted to enter the new coordinates of the markers. If you are operating Gepard in `WITEC_CONTROL` or `RENISHAW_CONTROL` mode, navigate your microscope stage to the markers and click `Read`. Depending on the calibration of your instruments, it might be necessary to invert one of more of the axes prior to transformation. If you notice very large residuals, please click `cancel` and try another combination. Notice that the warning about large residuals will appear regardless their values. Residuals around 10µm are usually to be expected.
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CAUTION: Due to a bug in the current version of Gepard, it is neccessary to additionally recalibrate the coordinate system, after Zeiss import. Please follow the description in the following section before proceeding.
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The transformation should now be complete and you should be able to continue with your measurement. To validate the transformation, activate the `Optical Scan mode` in the left toolbar and try to move your Raman stage by `Ctrl + click` on a particle on the Gepard image.
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# Recalibrate coordinate system
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This function correlates the real-world coordinates of the sample in the microscope to an existing Gepard project (e.g. if the sample was removed from the holder after image acquisition).
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In the Menu `Tools` choose `Recalibrate Coordinate System`. In the pop-up window click on the button `Add New Markers`.
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Use the control software of your microscope to navigate and focus on a characteristic feature of your sample. Then find the same feature on the GEpard image and click on it. A crosshair should have appeared on the image and the marker should be now listed in the window `Recalculate coordinate system`. Repeat this proccess at least twice. To correct the coordinates of a marker or to delete it, click on `Read Coordinates` or `Delete`, respectively.
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Transformation errors in the range of 10-20µm are to be expected. If the transformation errors at the bottom of window `Recalculate Coordinate System` are much larger, inversion of one or more axis might be necessary. To invert the axes, please tick the corresponding boxes at the top of the window.
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To save the transoformation, click the button `Save to Dataset`. |