Adding (‘+’) a position in the list allows the system to memorize its coordinates and focus (x,y,z), as well as the autofocus offsets. In a time-lapse acquisition, those positions will be re-visited at each time point. (3) Multi-Field Positions: That is to add multiple imaging positions in the sample. Both items have to be activated or inactivated together. (2) Focus Stabilization (hardware autofocusing): Start the function by ticking ‘Draft Stabilization Enabled’ (better to choose stabilization for ‘All Fields’) and by ticking ‘Draft Stabilization Mirror Inserted’. (b) Total time points or total time period of experiment: These two parameters are linked to each other. (a) Time interval: The time between the starts of two consecutive acquisitions For single point acquisition, it is ‘1’. Set up and acquire multi-dimensional acquisition (you may only need some of the following functions).(6) Tick ‘Protocol Channels’ and ‘Snap’ the images (step 2 of Figure 9). In the Camera section in Channel Manager, the cameras can be set to take multiple images ('Frame Averaging') and the images are averaged to generate a 'cleaner' image For the iXon EMCCD camera, 'Symmetrical Binning' can increase the camera sensitivity and image contrast, but at the cost of lower resolution of the images. In general, at least 3 times (the higher the better) difference in intensity between them is desired for publication quality images. Never set up those exposure parameters based on the display of the images Rather, you need to read the fluorescent intensities from the structure of interest and from the background by hovering the cursor over them. (5) Finding good exposure for each channel: Use ‘Active Channel’, start ‘Live’ mode (step 1 in Figure 9), adjust exposure time, laser intensity, and EM gain if the EMCCD camera is used (EM Gain at 200 is suggested)
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