In some cases, a fluorophore may turn ‘on’ and stay ‘on’ for multiple frames during data acquisition. In cases like this, treating each frame of the data separately, as is done in the localization phase, would lead to multiple appearances of the same fluorophore in the final reconstruction. The tracking phase uses an algorithm that identifies persistent fluorophores, and groups their appearances together into a unique track identifier. A fluorophore in one frame is considered to persist in subsequent frames if it is within a user-defined tracking search radius of its position in the previous frame. In some cases, a fluorophore may turn ‘off’ for a short time prior to turning on again. If the fluorophore is ‘off’ for fewer frames than the user-defined Fluorophore off time, the tracking algorithm will combine the two separate tracks into a single track, allowing the user to account for short-term blinking of the fluorophores in their sample. After persistent fluorophores are grouped into tracks, the track column of the *_LOC.txt or *LDC.txt file will be updated with the appropriate track identifier and saved as a new file with an *_LOCTracked.txt or *_LDCTracked.txt file extension.
If fiducial markers have been included in the sample, it is possible to use the tracking algorithm to measure the lateral drift at every frame during data acquisition. A fiducial marker is a long-lived fluorophore, often a fluorescent bead or gold nanoparticle, with tracks that span longer than a user-defined percentage of the total number of frames in the data set. Fiducial-marker-based drift correction can lead to the best drift correction results, especially in situations where the sample might be sparsely labeled or have drift on very short time scales.
Fiducial Drift Correction is done in two steps. The first step is to track all the fluorophores, identifying long-lived fluorophores that could be possible fiducial markers, and allowing the user to choose the best long-lived fluorophores to be used as fiducial markers. The second step is to correct fluorophore positions at each frame to account for lateral drift.
Note: It is not necessary to use fiducial markers to perform the tracking algorithm on an *_LOC.txt or *_LDC.txt file. Fiducial markers are only required for the fiducial drift correction. |