Single-Molecule FRET Microscopy Software

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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.
In the raw image, the donor emission channel is defined by the green ROI and the acceptor emission channel is defined by the red ROI. The image to the right is a color overlay of the two emission channels.
The emission channels are aligned when the peaks in each color channel are perfectly overlaid in the overlay image to the right.

Align emission channels manually

Drag in the two ROIs in the raw image to change their position and size. Move them until the peaks in each color channel in the overlay image to the right are overlaid.

Align emission channels automatically

Run auto-alignment by pressing the 'Auto-align ROIs' panel button just above the raw image. To perform well, the algorithm requires a fair number of peaks in each color channel as reference points (5-50).

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.
The Settings->Autoalign ROIs settings menu.

Fine-adjust alignment manually

Open the fine-tune window by pressing this panel button just above the raw image.
Use the arrows to change ROI positions one pixel at a time.

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.
Visual aids.
Revised 17.04.2023