Beads are automatically removed from iPALM data by first finding the beads in a rendered image and then filtering the localization list to remove localizations near a bead location.
Acknowledgments: The subfunctions bpass, cntrd, and pkfnd were adapted for MATLAB by Daniel Blair and Eric Dufresne from the IDL Particle Tracking software developed by David Grier, John Crocker, and Eric Weeks.
Bead removal takes as input an image and txt file generated by PeakSelector and outputs a new txt file with localizations corresponding to beads removed. This txt file can be reloaded into PeakSelector for further exploration and visualization of the data.
To use this approach:
- Load the data of interest into PeakSelector and ensure it is properly filtered. For example, set quality control settings such as sigma rtNPh < 0.06 or set bounds on the unwrapped z value.
- Export the PeakSelector data as an ASCII file with the option of xy values in pixels.
- Open the 'Cust. Tiff' menu and export the Total Raw Data as a tiff file with 133.33 nm per pixel.
- Fill in appropriate filenames for the ASCII and tiff files under the USER PARAMETERS section of
beadRemoval_v0_anisotropic.m. - Optionally adjust the parameters for bead removal:
rRemoveXandrRemoveYindicate the size of the region in pixels (133.33 nm/pixel) removed around each bead, in the X & Y directions respectively.rParticleindicates the approximate size of beads in the total raw data image, in pixels.beadThreshis a threshold for distinguishing beads from the background.
- Run
beadRemoval_v0_anisotropic.m. Figures will provide a check on the regions of data that were removed, and a new txt file will be generated without localizations from beads.
The output txt files can be reloaded into PeakSelctor through the menu option Import User ASCII by choosing xy coords in pixels, checking the box for headers, and inputting the values 0 to 48 for the data columns (see the file peakSelector_columnIndices for an easy list of values to copy and paste).