Aarhus University Seal

Align Donor and Acceptor Emission Channels

About emission channels in single-molecule FRET microscopy

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.

Align emission channels manually

Align emission channels automatically

When auto-alignment fails

When the auto-alignment fails these are the main possible causes:

  1. The initial positions of the two ROIs were not close enough for the algorithm to be able to detect the same peaks in each channel.
  2. The sample contains too many peaks.
  3. The sample contains too few peaks.

Possible solutions to each situation:

  1. Make a rough manual alignment of the ROI positions and then run auto-alignment again.
  2. Increase the number of peaks used for the auto-alignment. This setting is set in 'Settings->Auto align ROIs settings' or the panel menu item just above the raw image.
  3. Lower the number of peaks used for the auto-alignment.

Fine-adjust alignment manually

Visual aids

Adjust contrast

Adjust contrast in each emission channel using the contrast sliders above the raw and overlay images.

Define frame-interval of average image

Adjust the frame interval used for the average image by dragging the frame sliders above the raw and overlay images.

Define excitation channel of shown image

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.

Color blind?

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.