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[[_TOC_]]
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## Image acquisition
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The **Optical Scan** interface is used to set up the first set of the measurement, i.e. the image acquisition.
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[<img src="images/gepard-screenshots/optical_scan.jpg" width="600" />](images/gepard-screenshots/optical_scan.jpg)
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1. Define region of interest: Switch to the objective lens you plan on using for the measurement. Move the microscope stage to one of the outmost corners of the region you wish to scan and focus the microscope on the substrate (To facilitate precise focus on the substrate, you can use brightfield for this step). Under "Point coordinates" click `read` for the first point. Repeat for the other corners. You need the coordinates of at least three corners to set up image acquisition.
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However, depending on the method of substrate fit used later, we recommend reading the coordinates on at least five points (four corners and one additional point somewhere in the middle of the region) for more robust results.
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For better overview on where your current table position is, you can use `Image` to acquire an image without using it for fitting.
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1. Height fit mode
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Two methods for fitting the substrate surface are implemented in Gepard.
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`Level Fit` is sufficient for planar filters. `Triangulation` is more suitable it your substrate is uneven. To achieve a good fit with `Triangulation`, it is a good idea to record as many points of the substrate as possible.
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1. Focus levels
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The optical image will be generated by moving the sample table to all tile positions and acquiring several images at different focus positions. The focus positions are defined relative to the fitted substrate surface. Insert a number of focus steps and choose between `linear` and `custom` spacing.
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For linear spacing, the high and low values also need to be adjusted.
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More focus steps increase the level of detail and depth information used for the Raman/IR focus and for the calculate of particle height. However, the duration of image acquisition will also be affected. A good indicator for the step size is the focus depth of the lens used.
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1. Area Select
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Choose between `Rectangle` and `Circle`. To change the defined area, click and drag the boundary markers that are generated by the `Area select` button on the main window.
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1. Bright- or darkfield
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For microscopes with different brightfield and darkfield magnifications the current setting of the hardware must be adapted.
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1. Click `Run` to acquire the full image. Make sure that you select the brightfield or darkfield settings of the microscope before starting the process, depending on your preferences for light or dark background. Before the image tiling is started, the maximal z-shifts are computed for all tiles. Please note: for WITec systems only small movement ranges of plus and minus 100µm are allowed per default settings. If the specimen is tilted strongly or something went wrong in adjusting the fitting points, it may lead to large movement ranges. Use a central position as a last fitting point in this case to increase the possible range, because the WITec maximum range is alway relative to the current user defined position. **Important:** make sure, that the range that is displayed before the scan starts is acceptable with the current specimen and objective lens for all tile positions. Otherwise damage of the lens is possible! If you really know what you are doing, you can increase the maximum range in the WITec control settings.
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## Background subtraction
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Depending on your setup, your microscope image might include artefacts, e.g. vignetting, that are dusruptive in the full image.
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To counteract these effects, check the box `Enable Background Subtraction` and click `Show Background Manager Window`. Read one or more images of the background. The acquired images are averaged and the result will be subtracted from the sample images. Use the option to review the result on a 3x3 tiles image and correct as needed.
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## Single-pass mode
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This feature is still under development.
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It allows you to set up a fully automated measurement, in which image acquisition, particle detection and spectroscopic measurement are done automatically in one step, instead of setting up the particle detection after completion of the image acquisition. On the downside, the results of a fully automated, unsupervised particle detection is unlikely to be as good as those of the standard workflow. |