Lars BittrichThe Optical-Scan UI allows to define the area that will be scanned optically for generating the fullimage.
To setup the current measurement dimensions 3 to 5 points need to be selected manually for example by using the external controller supplied with WITec devices. Use the objective lens you plan on using for the measurement. All these points are selected by moving the table to the position and focussing the background of the specimen and click read at the corresponding point coordinate entry position in the UI. Using brightfield settings at the microscope allows for easy focus adjustment of the background. GEPARD will later make a surface fit with these three dimensional point data to determine the sample orientation. So using 5 points, i. e. four at each corner and one in the center yield most robust results. For better overview on where your current table position is, you can use Image to read an image without using is for later fitting. Please note: in the main window a full image will be computed and displayed using all positions read up to this point. For large magnifications and or large samples this might result in very large image dimensions, that can take very long time to compute and even might exhaust the main memory.
As a next step you can define the measurement area either in circle or rectangle form where the boundary of all currently saved fitting points are used. If you want to increase this area by a fixed amount you can define the Size increase value. It is also possible to manually move the red boundary markers that are generated by the Area select button to include for example small features at the boundary. For all boundary marker positions a new tiling (blue) is computed to cover all the selected are.
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 relative to the fitted surface orientation can be defined by the lower and upper limit and the number of steps. Next to standard linear spacing also a logarithmic and a custom spacing is possible. More focus steps increase the level of detail and depth information used to set the RAMAN laser focus later. However, also the measurement time to acquire the image will be increased. A good indicator for the step size is the focus depth of the lens currently used.
To cope with difficult lighting situations, a high dynamic range algorithm can be used. In this case the brightness will be adjusted for each focus position to three levels and a HDR image will be computed from all of these brightness settings. This will drastically increase measurement time and can also lead to background artifacts in dark field measurements.
For microscopes with different brightfield and darkfield magnifications the current setting of the hardware must be adapted.
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.