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Method Article
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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The authors declare no competing financial interests.
*These authors contributed equally to this work.
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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)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
W. E. Moerner
Moerner Laboratory, Department of Chemistry, Stanford University
Corresponding Author
Version 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
The authors declare no competing financial interests.
*These authors contributed equally to this work.
Loading...
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)