Making symmetric stacks in Mark & Find mode

This technical note describes what is probably the simplest way to take stacks at multiple positions using Mark & Find mode in the SP5 software. Using this procedure a symmetrical stack will be taken around each marked point. The instructions for this procedure are correct as of the 11th October 2016 but may need to be altered if software updates introduce changes. The procedure works on the SP5 but it should also be the same on the SPE3. If there are any differences in the procedure on the SPE3 then it will be noted.

Open Mark & Find and click on the Stage Configuration button indicated by arrow A in the image below. The Stage Configuration window will open. In the Stage Configuration window, click the Symmetric radio button indicated by arrow B.

Close the Stage Configuration window and focus on the specimen plane that you want to be in the middle of the stack. This might be different from the plane with the most fluorescence. For instance, an adherent cell may be more fluorescent near the cover-slip but the middle of the stack might be half way up the nucleus. It might be easiest to use a nuclear stain to locate the middle in this case but you will have to make sure the Begin and End of the stack are beyond the edges of the apical and basal membrane. Mark the position by clicking the button indicated by arrow C above. Make sure 'Same stack for all' is checked in the bottom left of the Mark & Find window.

Set up a Z series around the point you marked. The Begin and End of your series will automatically adjust so the marked point is the centre of the stack. Make sure the Z Step is set correctly. Mark more points; they will all automatically be the centre of Z stacks of the same range and Z Step as the first point.