To set up localization image refinement:
1. In the Localization Image Analysis window, navigate to the Tracking tab.
2. Activate the Do fluorophore tracking (refinement) check box.
3. In the Maximum localization precision (nm) field, enter the maximum localization precision in nanometers. Maximum localization precision indicates the largest amount of positional uncertainty a detected fluorophore may have to be used in the tracking algorithm.
4. Click the More Options button to display the More Tracking Options window.
5. In the Tracking search radius (nm) field, enter a search radius in nm for grouping detected fluorophores into one track of a single reappearing fluorophore. Smaller search radii will lead to higher certainty of proper grouping.
6. In the Fluorophore off time (frames) field, enter the maximum number of frames a fluorophore can be in the ‘off’ state before reappearing. This field is used to determine whether different fluorophore tracks are from a single fluorophore or from multiple fluorophores. A Fluorophore off time of 2 frames means that, to be considered as the same fluorophore, fluorophores may disappear for up to 2 frames before reappearing within 1 tracking search radius. This parameter allows the user to take into account short-term blinking of individual fluorophores.
7. Click the Done button to close the More Tracking Options window and return to the previous window.
8. If fiducial markers were included in the sample and the user would like to perform fiducial drift correction, select the Find fiducials as tracks spanning check box. In the Find fiducials as tracks spanning field, indicate the minimum number of frames (as a percentage of the total number of frames) that a fluorophore must be detected to be considered a fiducial. A fluorophore whose track spans less than this percent will not be considered a fiducial marker. The default value is 75%, but may need to be changed depending on how well the fiducials are localized throughout the data set.
9. Click the Run the Tracking button. If the Find fiducials as tracks spanning check box was not selected, the output file will be the same as the input, but with an updated track identifier column indicating fluorophore grouping (*_LOCTracked.txt or *_LDCTracked.txt).
10. If the Find fiducials as tracks spanning check box was selected, the Fiducial Drift Results window is displayed. The fiducial drift results is a graph presenting the lateral drift in x and y for up to ten long-lived fluorophores. Additionally, the raw data is also displayed, and next to each detected long-lived fluorophore is a number whose color corresponds to the color of the drift measurement in the fiducial drift results.
The presented long-lived fluorophores are those that are detected for greater than the percentage of frames chosen by the user, however not all of these fluorophores may give the best drift correction results. In some cases, some of the long-lived fluorophores may drift differently than the majority of the other long-lived fluorophores, and some long-lived fluorophores may come into the field of view intermittently. Therefore, it is important to choose the best long-lived fluorophores to be considered as fiducial markers. The best fiducial markers will show similar drift patterns and will be detected for the majority of the data set.
The user may select the best long-lived fluorophores to use as fiducial markers by clicking on the region of the legend that corresponds to the fiducial marker of choice. Selecting a fiducial marker to use increases the width of its drift measurement line, making it appear bold in comparison to the other lines representing long-lived fluorophores. It is possible to select between one and all of the long-lived fluorophores to be used as fiducial markers.
11. Once the selections are made, close the Fiducial Drift Results window. This will generate an output file that is the same as the input file, but with updated track identifiers in the Track column (*_LOCTracked.txt or *_LDCTracked.txt) and a new header column indicating which track identifier corresponds to a chosen fiducial marker.
12. If Fiducial Drift Correction is desired, and Step 10 has been completed, click the Run the Fiducial Drift Correction button to adjust the x and y coordinates to account for the drift measured from tracking the fiducial markers. If more than one fiducial marker has been chosen, the drift for all fiducial markers will be averaged, creating one drift measurement per frame. Running the fiducial drift correction will update the x- and y-coordinates and save a new file with all other inputs the same as the *_LOCTracked.txt or *_LDCTracked.txt input file. The new file will have the extension *_LDF.txt.
The output data for the tracking phase of a localization experiment is a file (*_LOCTracked.txt, *_LDCTracked.txt, or *_LDF.txt) with ten columns.
Note: The Track Identifier column now indicates a number corresponding to the track identifier for each fluorophore. The PSF Width column still contains ‘0’. |
If the user does not wish to do Fiducial Drift Correction, the output file produced will contain the same information as the input file with updated track identifiers in the Track Index column. Additionally, ‘Tracked’ is appended to the existing *_LOC.txt or *_LDC.txt filename. If the user performs Fiducial Drift Correction, the output file will be the same as the input file, but will contain the drift-corrected x- and y-coordinates and an updated Track column. The output file created will have the file extension *_LDF.txt for Localization Drift Corrected using Fiducial Markers.
Localization Phase Input Parameters
Setting Up Image Reconstruction