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Click the XY tab and click the Add button to add points as you move the stage around. Make sure the ‘Close active Shutter during Stage Movement’ at the bottom is ticked to reduce photobleaching and that the ‘Include Z’ box is ticked so the Z position is included along with the XY position. If the PFS is switched on the offset will be saved in the PFS column along with the X, Y and Z value at each position. It may be necessary to adjust the PFS offset at different positions, especially when marking points that are very distant to one another. If you mark a position with the PFS off and the focus changes when you turn the PFS on then the offset is different at that position. You can adjust it using the wheel on the PFS Offset Controller and then update the value in the software by clicking the arrow that appears next to the PFS column. X, Y and Z positions can be updated in the same way (see red arrows below). Finally, marked positions can be reordered using the up and down arrows in the toolbar and pressing the Optimize button will re-order the points so that the stage moves the shortest distance.
Click the Z tab to set the range and step size for Z series. Click on the ‘Defined by top bottom’ button to set a top and a bottom for the Z series. Set the Step size. Clicking the button next to the Step field will set the step to the optimum for the objective lens. For multi-position acquisitions the Z series must be Relative to compensate for the different Z positions of the marked points. Click the ‘Symmetric mode defined by range’ button and click Relative to make a symmetric z series around every point whose range is defined by the top and bottom you set above. ‘Asymmetric mode defined by range’ is more difficult to set up since you need to be able to type in the range above and below the Relative home position. Use the Order of Experiment button to define the order of acquisition for channels and Z slices.
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