In iSMS, the donor and acceptor emission channels are assumed to be located spatially next to each other within the raw image. There are no restrictions on relative positions of the two channels (left-right / up-down / etc.). The relative position of the two emission channels is defined by the two Regions-Of-Interest (ROIs) activated inside the raw image.
When the auto-alignment fails these are the main possible causes:
Possible solutions to each situation:
Adjust contrast in each emission channel using the contrast sliders above the raw and overlay images.
Adjust the frame interval used for the average image by dragging the frame sliders above the raw and overlay images.
If you run two-color alternating laser excitation (ALEX), you can choose which excitation channel to use for the average image. Navigate to the settings menu located at 'Settings->Avg. image settings'. In combination with the contrast sliders this is useful for a visual inspection of the laser spot profiles.
If you belong to the 10% of the male population being color blind, switch to a blue-yellow overlay view by ticking the 'View->Use colors for color blind' menu item.