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Method Article
Easy-DHPSF open-source software for three-dimensional localization of single molecules with precision beyond the optical diffraction limit
W. E. Moerner
Moerner Laboratory, Department of Chemistry, Stanford University
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Automated processing of double-helix (DH) microscope images of single molecules (SMs) streamlines the protocol required to obtain super-resolved three-dimensional (3D) reconstructions of ultrastructures in biological samples by single-molecule active control microscopy. Here, we present a "suite of MATLAB subroutines":https://code.google.com/p/easy-dhpsf/, bundled with an easy-to-use "graphical user interface (GUI)":#f0, that facilitates 3D localization of single emitters (e.g. SMs, fluorescent beads, or quantum dots) with precisions of tens of nanometers in multi-frame movies acquired using a wide-field DH epifluorescence microscope. The algorithmic approach is based upon template matching for SM recognition and least-squares fitting for 3D position measurement, both of which are computationally expedient and precise. Overlapping images of SMs are ignored, and the precision of least-squares fitting is not as high as maximum likelihood-based methods. However, once calibrated, the algorithm can fit 15-30 molecules per second on a 3 GHz Intel Core 2 Duo workstation, thereby producing a 3D super-resolution reconstruction of 100,000 molecules over a 20×20×2 μm field of view (processing 128×128 pixels × 20000 frames) in "75 min":#time_taken.
Keywords
Super-resolution, superresolution, localization microscopy, single molecule, fluorescence, PALM, STORM, fPALM, SMACM, double helix, DHPSF, three-dimensional imaging, 3D, template matching, image correlation, nonlinear least-squares, wavelet, MATLAB, graphical user interface, GUI, open source
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Associated Publications
PNAS Plus: Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus
by M. D. Lew, S. F. Lee, J. L. Ptacin, +4
Proceedings Of The National Academy Of Sciences (18 February, 2013)
The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision
by Hsiao-lu D. Lee, Steffen J. Sahl, Matthew D. Lew, +1
Applied Physics Letters (18 February, 2013)
Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in 3D
by Andreas Gahlmann, Jerod Louis Ptacin, Ginni Grover, +7
Nano Letters (18 February, 2013)
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This is a preprint. It has not completed peer review.
Method Article
Easy-DHPSF open-source software for three-dimensional localization of single molecules with precision beyond the optical diffraction limit
W. E. Moerner
Moerner Laboratory, Department of Chemistry, Stanford University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 25 Feb, 2013
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Automated processing of double-helix (DH) microscope images of single molecules (SMs) streamlines the protocol required to obtain super-resolved three-dimensional (3D) reconstructions of ultrastructures in biological samples by single-molecule active control microscopy. Here, we present a "suite of MATLAB subroutines":https://code.google.com/p/easy-dhpsf/, bundled with an easy-to-use "graphical user interface (GUI)":#f0, that facilitates 3D localization of single emitters (e.g. SMs, fluorescent beads, or quantum dots) with precisions of tens of nanometers in multi-frame movies acquired using a wide-field DH epifluorescence microscope. The algorithmic approach is based upon template matching for SM recognition and least-squares fitting for 3D position measurement, both of which are computationally expedient and precise. Overlapping images of SMs are ignored, and the precision of least-squares fitting is not as high as maximum likelihood-based methods. However, once calibrated, the algorithm can fit 15-30 molecules per second on a 3 GHz Intel Core 2 Duo workstation, thereby producing a 3D super-resolution reconstruction of 100,000 molecules over a 20×20×2 μm field of view (processing 128×128 pixels × 20000 frames) in "75 min":#time_taken.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
Figure 15
Figure 16
Associated Publications
PNAS Plus: Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus
by M. D. Lew, S. F. Lee, J. L. Ptacin, +4
Proceedings Of The National Academy Of Sciences (18 February, 2013)
The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision
by Hsiao-lu D. Lee, Steffen J. Sahl, Matthew D. Lew, +1
Applied Physics Letters (18 February, 2013)
Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in 3D
by Andreas Gahlmann, Jerod Louis Ptacin, Ginni Grover, +7
Nano Letters (18 February, 2013)