Channel registration

This function corrects image aberrations differently affecting SMLM datasets taken in separate fluorescence emission channels. The correction procedure is divided into two steps. First, a local wheighted mean transformation (LWM) is calculated from two images showing fluorescent beads in both emission channels. Important: Take fluorescent bead reference images periodically to ensure proper image registration. During the Drift correction, fiducials are used to track and correct for sample drift during the STORM acquisition. Fiducials visible in both emission channels are then used for the second correction step where the drift corrected fiducial centers are matched using a rigid translation.

Select Image Registration from the SPARTAN menu.

1. Open both reference images (example data) taken in both emission channels

The two images are shown in the center display area.

2. Select the approximate particle diameter and apply band pass filter. The image display will be updated accordingly.
3. Localize the particles (filter by selecting a brightness and diameter threshold for both channels). This will start a two-step localization routine. First particle candidates will be selected according to the local brightness, then their centers are localized with sub-pixel precision.
4. Show localization histogram (radius of gyration, intenisty) to filter bad localizations. Important: This filter makes sure that only individual beads are selected.

The identified and filtered localizations are displayed in the top right panel.

5. Repeat steps 2-4 until you see a satisfying selection of bead pairs
6. Calculate the local weighted mean (LWM) transformation between both channels.

The target registration error (TRE) will be calculated and shown as a result. Typically around 10 nm.

6. Save the transformation