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Check the Sequential checkbox to make sure that the different colour channels in your images are acquired sequentially rather than simultaneously. Sequential imaging is slower than simultaneous but reduces crosstalk when using fluorophores with overlapping excitation or emission spectra.
You can define which channels are scanned simultaneously and which are scanned sequentially by dragging them into different Groups. In the example below Alexa Fluor 488 and Alexa Fluor 568 are in different groups and will be scanned sequentially. If I was also imaging Alexa Fluor 647 I could have that in a third group or I could shorten the scan time by dragging it into Group 1 to be scanned simultaneously with Alexa Fluor 488. It is possible to scan a Live View in sequential mode but when Frame sequential is selected only the currently selected channel will be refreshed.
Z series
A Z-stack can be configured in the Acquisition Settings panel. If you want to reset the current focus position to zero you can do that by pressing Set 0. Focus on the plane where you would like to start the Z-stack and press Set under Start, then focus on the last plane of the stack and press Set under End.
Warning If you want to reset the focus position with Set 0 do it before you set the Start and End positions, otherwise the Z series will be many microns out of focus.
Set a Step size in μm by typing in a value or press Op. for the microscope to set the optimal step size for the objective lens currently being used. If the Z-stack menu is hidden just double-click on Microscope to display it. Click Depth beneath the XY button in Image Acquisition Control. The XY button starts the acquisition, but if Depth is not clicked then only a single XY plane (no Z) will be scanned. The Lambda, Time, etc. buttons should be activated for spectral scans, time series and other combinations.
- When the Z series is finished you must press Series Done (below) to open a window displaying the Z-stack, which you can then save. Alternatively you can press Append Next to acquire another Z-stack and append it to the previous one, but this is not likely to be a common practice.
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