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.

Features * Double-Gaussian estimator is calibrated via an axial scan of bright immobile fluorescent emitters (e.g. beads). * Template-matching and double-helix recognition algorithm is calibrated via setting an image correlation threshold against single-molecule data. * Tiff stacks of SM images are analyzed using template matching followed by double-Gaussian fitting to extract estimates of the molecule positions. * The motion/drift of fiduciaries (e.g. fluorescent beads) during an experiment can tracked and removed form SM localization data. * 3D localization data can be exported to .csv format for post-processing and visualization. * Basic 3D scatterplot and 2D z-projection histogram visualizations are included.

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Getting started

System requirements

MATLAB and its associated image processing, optimization, statistics, and wavelet toolboxes are required. The code has been tested with MATLAB R2011b and R2012b. No known issues exist with other versions of MATLAB, but some built-in functions (e.g. fft2, imread) may exhibit degraded performance in older versions of MATLAB.

Downloading and installing

The source code can be downloaded via the instructions below. No installation is needed; simply place the source code in your MATLAB path and run easy_dhpsf from the MATLAB command line. As usual, read the documentation first!

Stable version

Note: Google has recently disabled the "downloads" functionality of Google code. Please use our Mercurial repository to keep your software up to date.

A stable version of the source code is available as a zip archive. Download it from the featured downloads list at left.

• Version 1.02: Fixed bugs in f_fitSMs and f_concatSMfits

• Version 1.01: Streamlined f_calDHPSF

• Version 1.0: Initial build

Latest version

Please use Mercurial to keep up to date with the latest features and bug-fixes. hg clone https://your-google-username@code.google.com/p/easy-dhpsf/ A great Windows client for Mercurial is TortoiseHg.

Documentation

There are two versions of the documentation available. * Lew, M. D.*, von Diezmann, A. R. S.*, and Moerner, W. E. “Easy-DHPSF open-source software for three-dimensional localization of single molecules with precision beyond the optical diffraction limit” Protocol Exchange (2013). DOI: 10.1038/protex.2013.026 * PDF located in the featured downloads list at left

Sample single-molecule DH-PSF data

The sample files entitled "MT_sample_data" can be found in the Downloads section. This data was published in:

Lee, H. D.*, Sahl, S. J.*, Lew, M. D. & Moerner, W. E. The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision. App. Phys. Lett. 100, 153701 (2012). [link]

Program history and development team

This code grew out of a desire to automate the fitting process for DH microscope data as a "Digital Image Processing" class project. Since then, features have been added and bugs have been fixed to make it ready for us by others with MATLAB experience.

• Version 1.0, 2012-present: Matthew D. Lew, Alexander R. S. von Diezmann, Andreas Gahlmann, Mikael P. Backlund, W. E. Moerner

  Featured in:

  1. Gahlmann, A. et al. Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in 3D. Nano Lett. (2013). [link]

• Version 0.5, Summer 2010-2011: Matthew D. Lew, Steven F. Lee, Steffen J. Sahl, Alex Chang, W. E. Moerner

  Featured in:

  1. Lew, M. D.*, Lee S. F.*, et al. Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus. Proc. Natl. Acad. Sci. USA 108, E1102-E1110 (2011). [link]
  2. Lee, H. D.*, Sahl, S. J.*, Lew, M. D. & Moerner, W. E. The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision. App. Phys. Lett. 100, 153701 (2012). [link]

• Version 0.1, Spring 2010: Matthew D. Lew and Scott S. Hsieh

  EE 368 Digital Image Processing class project