Script to find double-dots and extract molecular coordinates from DHPSF microscopy data, pre-processed by extracting single localisations.
Authors: Aleks Ponjavic, University of Leeds; Aleksandra Jartseva, University of Cambridge
To run this, you need both the main script DHPSFUAngle.m and the auxiliary function calibAngle.m in your working directory. Open DHPSFUAngle.m, edit the necessary parameters, and run it.
- Data for analysis in the form of a text file containing a list of single localisations. To enhance batch processing, the code loops through all files with the correct extension in the supplied folder.
- Coordinates in pixels.
- The recommended fitting software is GDSC SMLM PeakFit.
- Calibration file, similarly formatted.
- Must contain a single fiducial, imaged at equally-spaced z-coordinates in the desired range. E.g. it could have been imaged every 50 nm from -2 um to 2 um in z. There must be one frame per z-coordinate, i.e. there must be a one-step movement between each frame.
- Pixel size (in nm).
- Precision cutoff, above which the Peak Fit localisations will be filtered out.
- The step size in z in the calibration sequence.
- Filtering parameters: the initial values for filtering out pairs of localisations that are too far and too close, and the allowed deviation in the distance and intensity ratio, compared to the calibration.
Output will be written into the specified folder in the ViSP .3d format: x y z intensity frame, tab-separated.