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GUI for fitting 3D single-molecule images using the double-helix PSF
This package of MATLAB routines with accompanying GUI allows scientific users to measure the 3D location of single molecules when using the double-helix point spread function (DH-PSF) widefield microscope. Version 2 also includes code that facilitates combining localization data from two spectral channels using a locally-weighted quadratic 3D registration function
Features
- Double-Gaussian estimator is calibrated via an axial scan of bright immobile fluorescent emitters (e.g. beads)
- DH localization is performed through template matching and subsequent double Gaussian fitting
- Tiff stacks of SM images are analyzed using template matching followed by double-Gaussian fitting to extract estimates of the molecule positions
- Two spectral channels are registered by generating a locally-weighted quadratic mapping function from control point pairs
...Perform a phase retrieval algorithm based on maximum likelihood estimation (MLE) of a phase aberration term which is added to the theoretical pupil function of the imaging system.
3. Use the phase-retrieved pupil function to perform single-emitter localization.
Accompanying publication available here: https://doi.org/10.1364/OE.25.007945