Abstract

Single-molecule orientation measurements provide unparalleled insight into a multitude of biological and polymeric systems. We report a simple, high-throughput technique for measuring the azimuthal orientations and rotational dynamics of single fluorescent molecules, which is compatible with localization microscopy. Our method involves modulating the polarization of an excitation laser and analyzing the corresponding intensities emitted by single dye molecules and their modulation amplitudes. To demonstrate our approach, we use intercalating and groove-binding dyes to obtain super-resolved images of stretched DNA strands through binding-induced turn-on of fluorescence. By combining our image data with thousands of dye molecule orientation measurements, we develop a means of probing the structure of individual DNA strands, while also characterizing dye-DNA interactions. This approach may hold promise as a method for monitoring DNA conformation changes resulting from DNA-binding proteins.

© 2016 Optical Society of America

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References

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2016 (2)

C. A. Cruz, H. Shaban Ahmed, A. Kress, N. Bertaux, S. Monneret, M. Mavrakis, J. Savatier, and S. Brasselet, “Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy,” Proc. Natl. Acad. Sci. USA 113, E820–E828 (2016).
[Crossref]

R. Börner, N. Ehrlich, J. Hohlbein, and C. G. Hübner, “Single molecule 3D orientation in time and space: a 6D dynamic study on fluorescently labeled lipid membranes,” J. Fluoresc., 26, 963–975 (2016).

2015 (11)

K. I. Mortensen, J. Sung, H. Flyvbjerg, and J. A. Spudich, “Optimized measurements of separations and angles between intra-molecular fluorescent markers,” Nat. Commun. 6, 8621 (2015).
[Crossref]

M. Hashimoto, K. Yoshiki, M. Kurihara, N. Hashimoto, and T. Araki, “Orientation detection of a single molecule using pupil filter with electrically controllable polarization pattern,” Opt. Rev., 22, 875–881 (2015).

W. E. Moerner, “Nobel lecture: single-molecule spectroscopy, imaging, and photocontrol: foundations for super-resolution microscopy,” Rev. Mod. Phys. 87, 1183–1212 (2015).
[Crossref]

A. von Diezmann, M. Y. Lee, M. D. Lew, and W. Moerner, “Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy,” Optica 2, 985–993 (2015).
[Crossref]

L. Carlini, S. J. Holden, K. M. Douglass, and S. Manley, “Correction of a depth-dependent lateral distortion in 3D super-resolution imaging,” Plos One 10, e0142949 (2015).
[Crossref]

S. Stallinga, “Effect of rotational diffusion in an orientational potential well on the point spread function of electric dipole emitters,” J. Opt. Soc. Am. A 32, 213–223 (2015).
[Crossref]

A. S. Backer and W. E. Moerner, “Determining the rotational mobility of a single molecule from a single image: a numerical study,” Opt. Express 23, 4255–4276 (2015).
[Crossref]

N. Karedla, S. C. Stein, D. Hähnel, I. Gregor, A. Chizhik, and J. Enderlein, “Simultaneous measurement of the three-dimensional orientation of excitation and emission dipoles,” Phys. Rev. Lett. 115, 173002 (2015).
[Crossref]

S. Abrahamsson, M. McQuilken, S. B. Mehta, A. Verma, J. Larsch, R. Ilic, R. Heintzmann, C. I. Bargmann, A. S. Gladfelter, and R. Oldenbourg, “MultiFocus polarization microscope (MF-PolScope) for 3D polarization imaging of up to 25 focal planes simultaneously,” Opt. Express 23, 7734–7754 (2015).
[Crossref]

A. S. Biebricher, I. Heller, R. F. H. Roijmans, T. P. Hoekstra, and E. J. G. Peterman, “The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics,” Nat. Commun. 6, 7304 (2015).
[Crossref]

H. Miller, Z. Zhou, A. Wollman, and M. C. Leake, “Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes,” Methods 88, 81–88 (2015).
[Crossref]

2014 (3)

N. Hafi, M. Grunwald, L. S. van den Heuvel, T. Aspelmeier, J. Chen, M. Zagrebelsky, O. M. Schütte, C. Steinem, M. Korte, A. Munk, and P. J. Walla, “Fluorescence nanoscopy by polarization modulation and polarization angle narrowing,” Nat. Methods 11, 579–584 (2014).
[Crossref]

M. D. Lew and W. E. Moerner, “Azimuthal polarization filtering for accurate, precise, and robust single-molecule localization microscopy,” Nano Lett. 14, 6407–6413 (2014).
[Crossref]

S. Bakshi, H. Choi, N. Rangarajan, K. J. Barns, B. P. Bratton, and J. C. Weisshaar, “Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack,” Appl. Environ. Microbiol. 80, 4977–4986 (2014).
[Crossref]

2013 (3)

A. Kress, X. Wang, H. Ranchon, J. Savatier, H. Rigneault, P. Ferrand, and S. Brasselet, “Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy,” Biophys. J. 105, 127–136 (2013).
[Crossref]

M. D. Lew, M. P. Backlund, and W. E. Moerner, “Rotational mobility of single molecules affects localization accuracy in super-resolution fluorescence microscopy,” Nano Lett. 13, 3967–3972 (2013).
[Crossref]

A. S. Backer, M. P. Backlund, M. D. Lew, and W. E. Moerner, “Single-molecule orientation measurements with a quadrated pupil,” Opt. Lett. 38, 1521–1523 (2013).
[Crossref]

2012 (2)

S. Stallinga and B. Rieger, “Position and orientation estimation of fixed dipole emitters using an effective Hermite point spread function model,” Opt. Express 20, 5896–5921 (2012).
[Crossref]

M. P. Backlund, M. D. Lew, A. S. Backer, S. J. Sahl, G. Grover, A. Agrawal, R. Piestun, and W. E. Moerner, “Simultaneous, accurate measurement of the 3D position and orientation of single molecules,” Proc. Natl. Acad. Sci. USA 109, 19087–19092 (2012).
[Crossref]

2011 (3)

A. I. Chizhik, A. M. Chizhik, A. Huss, R. Jäger, and A. J. Meixner, “Nanoscale probing of dielectric interfaces with single-molecule excitation patterns and radially polarized illumination,” J. Phys. Chem. Lett. 2, 2152–2157 (2011).
[Crossref]

J. Engelhardt, J. Keller, P. Hoyer, M. Reuss, T. Staudt, and S. W. Hell, “Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy,” Nano Lett. 11, 209–213 (2011).
[Crossref]

I. Schoen, J. Ries, E. Klotzsch, H. Ewers, and V. Vogel, “Binding-activated localization microscopy of DNA structures,” Nano Lett. 11, 4008–4011 (2011).
[Crossref]

2010 (4)

S. Stallinga and B. Rieger, “Accuracy of the Gaussian point spread function model in 2D localization microscopy,” Opt. Express 18, 24461–24476 (2010).
[Crossref]

R. Jungmann, C. Steinhauer, M. Scheible, A. Kuzyk, P. Tinnefeld, and F. C. Simmel, “Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami,” Nano Lett. 10, 4756–4761 (2010).
[Crossref]

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466, 647–651 (2010).
[Crossref]

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, “Optimized localization analysis for single-molecule tracking and super-resolution microscopy,” Nat. Methods 7, 377–381 (2010).
[Crossref]

2009 (2)

2008 (4)

T. Fazio, M. L. Visnapuu, S. Wind, and E. C. Greene, “DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging,” Langmuir 24, 10524–10531 (2008).
[Crossref]

C. Y. Lu and D. A. Van den Bout, “Analysis of orientation dynamics of single fluorophore trajectories from three-angle polarization experiments,” J. Chem. Phys. 128, 244501 (2008).
[Crossref]

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5, 1027–1030 (2008).
[Crossref]

I. Testa, A. Schönle, C. von Middendorff, C. Geisler, R. Medda, C. A. Wurm, A. C. Stiel, S. Jakobs, M. Bossi, C. Eggeling, S. W. Hell, and A. Egner, “Nanoscale separation of molecular species based on their rotational mobility,” Opt. Express 16, 21093–21104 (2008).
[Crossref]

2006 (6)

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91, 4258–4272 (2006).
[Crossref]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, 793–796 (2006).
[Crossref]

A. Sharonov and R. M. Hochstrasser, “Wide-field subdiffraction imaging by accumulated binding of diffusing probes,” Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).
[Crossref]

E. Toprak, J. Enderlein, S. Syed, S. A. McKinney, R. G. Petschek, T. Ha, Y. E. Goldman, and P. R. Selvin, “Defocused orientation and position imaging (DOPI) of myosin V,” Proc. Natl. Acad. Sci. USA 103, 6495–6499 (2006).
[Crossref]

J. Enderlein, E. Toprak, and P. R. Selvin, “Polarization effect on position accuracy of fluorophore localization,” Opt. Express 14, 8111–8120 (2006).
[Crossref]

2005 (4)

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, “Measurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy,” Biophys. J. 89, 1261–1271 (2005).
[Crossref]

M. E. Quinlan, J. N. Forkey, and Y. E. Goldman, “Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy,” Biophys. J. 89, 1132–1142 (2005).
[Crossref]

S. A. Rosenberg, M. E. Quinlan, J. N. Forkey, and Y. E. Goldman, “Rotational motions of macro-molecules by single-molecule fluorescence microscopy,” Acc. Chem. Res. 38, 583–593 (2005).
[Crossref]

J. Hohlbein and C. G. Hubner, “Simple scheme for rapid three-dimensional orientation determination of the emission dipole of single molecules,” Appl. Phys. Lett. 86, 121104 (2005).
[Crossref]

2004 (1)

D. Patra, I. Gregor, and J. Enderlein, “Image analysis of defocused single-molecule images for three-dimensional molecule orientation studies,” J. Phys. Chem. A 108, 6836–6841 (2004).
[Crossref]

2003 (2)

M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B 20, 554–559 (2003).
[Crossref]

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. Corrie, and Y. E. Goldman, “Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization,” Nature 422, 399–404 (2003).
[Crossref]

2002 (2)

N. B. Bowden, K. A. Willets, W. E. Moerner, and R. M. Waymouth, “Synthesis of fluorescently labeled polymers and their use in single-molecule imaging,” Macromolecules 35, 8122–8125 (2002).
[Crossref]

L. H. Hurley, “DNA and its associated processes as targets for cancer therapy,” Nat. Rev. Cancer 2, 188–200 (2002).
[Crossref]

2001 (4)

J. T. Fourkas, “Rapid determination of the three-dimensional orientation of single molecules,” Opt. Lett. 26, 211–213 (2001).
[Crossref]

K. D. Weston and L. S. Goldner, “Orientation imaging and reorientation dynamics of single dye molecules,” J. Phys. Chem. B 105, 3453–3462 (2001).
[Crossref]

H. Sosa, E. J. G. Peterman, W. E. Moerner, and L. S. B. Goldstein, “ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy,” Nat. Struct. Biol. 8, 540–544 (2001).
[Crossref]

E. J. G. Peterman, H. Sosa, L. S. B. Goldstein, and W. E. Moerner, “Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules,” Biophys. J. 81, 2851–2863 (2001).
[Crossref]

2000 (3)

D. Hu, J. Yu, K. Wong, B. Bagchi, P. J. Rossky, and P. F. Barbara, “Collapse of stiff conjugated polymers with chemical defects into ordered cylindrical conformations,” Nature 405, 1030–1033 (2000).
[Crossref]

X. Yan, R. C. Habbersett, J. M. Cordek, J. P. Nolan, T. M. Yoshida, J. H. Jett, and B. L. Marrone, “Development of a mechanism-based DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry,” Anal. Biochem. 286, 138–148 (2000).
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B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85, 4482–4485 (2000).
[Crossref]

1999 (1)

M. L. Bennink, O. D. Schärer, R. Kanaar, K. Sakata-Sogawa, J. M. Schins, J. S. Kanger, B. G. Grooth, and J. Greve, “Single-molecule manipulation of double-stranded DNA using optical tweezers: Interaction studies of DNA with RecA and YOYO-1,” Cytometry 36, 200–208 (1999).
[Crossref]

1998 (2)

T. Ha, J. Glass, T. Enderle, D. S. Chemla, and S. Weiss, “Hindered rotational diffusion and rotational jumps of single molecules,” Phys. Rev. Lett. 80, 2093–2096 (1998).
[Crossref]

R. M. Dickson, D. J. Norris, and W. E. Moerner, “Simultaneous imaging of individual molecules aligned both parallel and perpendicular to the optic axis,” Phys. Rev. Lett. 81, 5322–5325 (1998).
[Crossref]

1997 (1)

X. Michalet, R. Ekong, F. Fougerousse, S. Rousseaux, C. Schurra, N. Hornigold, M. Slegtenhorst, J. Wolfe, S. Povey, J. S. Beckmann, and A. Bensimon, “Dynamic molecular combing: stretching the whole human genome for high-resolution studies,” Science 277, 1518–1523 (1997).
[Crossref]

1996 (1)

T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77, 3979–3982 (1996).
[Crossref]

1995 (1)

D. Bensimon, A. J. Simon, V. Croquette, and A. Bensimon, “Stretching DNA with a receding meniscus: experiments and models,” Phys. Rev. Lett. 74, 4754–4757 (1995).
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Abrahamsson, S.

Agrawal, A.

M. P. Backlund, M. D. Lew, A. S. Backer, S. J. Sahl, G. Grover, A. Agrawal, R. Piestun, and W. E. Moerner, “Simultaneous, accurate measurement of the 3D position and orientation of single molecules,” Proc. Natl. Acad. Sci. USA 109, 19087–19092 (2012).
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Aguet, F.

Araki, T.

M. Hashimoto, K. Yoshiki, M. Kurihara, N. Hashimoto, and T. Araki, “Orientation detection of a single molecule using pupil filter with electrically controllable polarization pattern,” Opt. Rev., 22, 875–881 (2015).

Arbabi, A.

M. P. Backlund, A. Arbabi, P. N. Petrov, E. Arbabi, S. Saurabh, A. Faraon, and W. E. Moerner (2016), doi: 10.1038/NPHOTON.2016.93 (to be published).

Arbabi, E.

M. P. Backlund, A. Arbabi, P. N. Petrov, E. Arbabi, S. Saurabh, A. Faraon, and W. E. Moerner (2016), doi: 10.1038/NPHOTON.2016.93 (to be published).

Aspelmeier, T.

N. Hafi, M. Grunwald, L. S. van den Heuvel, T. Aspelmeier, J. Chen, M. Zagrebelsky, O. M. Schütte, C. Steinem, M. Korte, A. Munk, and P. J. Walla, “Fluorescence nanoscopy by polarization modulation and polarization angle narrowing,” Nat. Methods 11, 579–584 (2014).
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Backer, A. S.

A. S. Backer and W. E. Moerner, “Determining the rotational mobility of a single molecule from a single image: a numerical study,” Opt. Express 23, 4255–4276 (2015).
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A. S. Backer, M. P. Backlund, M. D. Lew, and W. E. Moerner, “Single-molecule orientation measurements with a quadrated pupil,” Opt. Lett. 38, 1521–1523 (2013).
[Crossref]

M. P. Backlund, M. D. Lew, A. S. Backer, S. J. Sahl, G. Grover, A. Agrawal, R. Piestun, and W. E. Moerner, “Simultaneous, accurate measurement of the 3D position and orientation of single molecules,” Proc. Natl. Acad. Sci. USA 109, 19087–19092 (2012).
[Crossref]

Backlund, M. P.

M. D. Lew, M. P. Backlund, and W. E. Moerner, “Rotational mobility of single molecules affects localization accuracy in super-resolution fluorescence microscopy,” Nano Lett. 13, 3967–3972 (2013).
[Crossref]

A. S. Backer, M. P. Backlund, M. D. Lew, and W. E. Moerner, “Single-molecule orientation measurements with a quadrated pupil,” Opt. Lett. 38, 1521–1523 (2013).
[Crossref]

M. P. Backlund, M. D. Lew, A. S. Backer, S. J. Sahl, G. Grover, A. Agrawal, R. Piestun, and W. E. Moerner, “Simultaneous, accurate measurement of the 3D position and orientation of single molecules,” Proc. Natl. Acad. Sci. USA 109, 19087–19092 (2012).
[Crossref]

M. P. Backlund, A. Arbabi, P. N. Petrov, E. Arbabi, S. Saurabh, A. Faraon, and W. E. Moerner (2016), doi: 10.1038/NPHOTON.2016.93 (to be published).

Bagchi, B.

D. Hu, J. Yu, K. Wong, B. Bagchi, P. J. Rossky, and P. F. Barbara, “Collapse of stiff conjugated polymers with chemical defects into ordered cylindrical conformations,” Nature 405, 1030–1033 (2000).
[Crossref]

Bakshi, S.

S. Bakshi, H. Choi, N. Rangarajan, K. J. Barns, B. P. Bratton, and J. C. Weisshaar, “Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack,” Appl. Environ. Microbiol. 80, 4977–4986 (2014).
[Crossref]

Barbara, P. F.

D. Hu, J. Yu, K. Wong, B. Bagchi, P. J. Rossky, and P. F. Barbara, “Collapse of stiff conjugated polymers with chemical defects into ordered cylindrical conformations,” Nature 405, 1030–1033 (2000).
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Bargmann, C. I.

Barns, K. J.

S. Bakshi, H. Choi, N. Rangarajan, K. J. Barns, B. P. Bratton, and J. C. Weisshaar, “Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack,” Appl. Environ. Microbiol. 80, 4977–4986 (2014).
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M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, 793–796 (2006).
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X. Michalet, R. Ekong, F. Fougerousse, S. Rousseaux, C. Schurra, N. Hornigold, M. Slegtenhorst, J. Wolfe, S. Povey, J. S. Beckmann, and A. Bensimon, “Dynamic molecular combing: stretching the whole human genome for high-resolution studies,” Science 277, 1518–1523 (1997).
[Crossref]

Bennink, M. L.

M. L. Bennink, O. D. Schärer, R. Kanaar, K. Sakata-Sogawa, J. M. Schins, J. S. Kanger, B. G. Grooth, and J. Greve, “Single-molecule manipulation of double-stranded DNA using optical tweezers: Interaction studies of DNA with RecA and YOYO-1,” Cytometry 36, 200–208 (1999).
[Crossref]

Bensimon, A.

X. Michalet, R. Ekong, F. Fougerousse, S. Rousseaux, C. Schurra, N. Hornigold, M. Slegtenhorst, J. Wolfe, S. Povey, J. S. Beckmann, and A. Bensimon, “Dynamic molecular combing: stretching the whole human genome for high-resolution studies,” Science 277, 1518–1523 (1997).
[Crossref]

D. Bensimon, A. J. Simon, V. Croquette, and A. Bensimon, “Stretching DNA with a receding meniscus: experiments and models,” Phys. Rev. Lett. 74, 4754–4757 (1995).
[Crossref]

Bensimon, D.

D. Bensimon, A. J. Simon, V. Croquette, and A. Bensimon, “Stretching DNA with a receding meniscus: experiments and models,” Phys. Rev. Lett. 74, 4754–4757 (1995).
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Bertaux, N.

C. A. Cruz, H. Shaban Ahmed, A. Kress, N. Bertaux, S. Monneret, M. Mavrakis, J. Savatier, and S. Brasselet, “Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy,” Proc. Natl. Acad. Sci. USA 113, E820–E828 (2016).
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Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
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Biebricher, A. S.

A. S. Biebricher, I. Heller, R. F. H. Roijmans, T. P. Hoekstra, and E. J. G. Peterman, “The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics,” Nat. Commun. 6, 7304 (2015).
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Böhmer, M.

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref]

Börner, R.

R. Börner, N. Ehrlich, J. Hohlbein, and C. G. Hübner, “Single molecule 3D orientation in time and space: a 6D dynamic study on fluorescently labeled lipid membranes,” J. Fluoresc., 26, 963–975 (2016).

Bossi, M.

Bowden, N. B.

N. B. Bowden, K. A. Willets, W. E. Moerner, and R. M. Waymouth, “Synthesis of fluorescently labeled polymers and their use in single-molecule imaging,” Macromolecules 35, 8122–8125 (2002).
[Crossref]

Brasselet, S.

C. A. Cruz, H. Shaban Ahmed, A. Kress, N. Bertaux, S. Monneret, M. Mavrakis, J. Savatier, and S. Brasselet, “Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy,” Proc. Natl. Acad. Sci. USA 113, E820–E828 (2016).
[Crossref]

A. Kress, X. Wang, H. Ranchon, J. Savatier, H. Rigneault, P. Ferrand, and S. Brasselet, “Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy,” Biophys. J. 105, 127–136 (2013).
[Crossref]

Bratton, B. P.

S. Bakshi, H. Choi, N. Rangarajan, K. J. Barns, B. P. Bratton, and J. C. Weisshaar, “Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack,” Appl. Environ. Microbiol. 80, 4977–4986 (2014).
[Crossref]

Carlini, L.

L. Carlini, S. J. Holden, K. M. Douglass, and S. Manley, “Correction of a depth-dependent lateral distortion in 3D super-resolution imaging,” Plos One 10, e0142949 (2015).
[Crossref]

Chemla, D. S.

T. Ha, J. Glass, T. Enderle, D. S. Chemla, and S. Weiss, “Hindered rotational diffusion and rotational jumps of single molecules,” Phys. Rev. Lett. 80, 2093–2096 (1998).
[Crossref]

T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77, 3979–3982 (1996).
[Crossref]

Chen, J.

N. Hafi, M. Grunwald, L. S. van den Heuvel, T. Aspelmeier, J. Chen, M. Zagrebelsky, O. M. Schütte, C. Steinem, M. Korte, A. Munk, and P. J. Walla, “Fluorescence nanoscopy by polarization modulation and polarization angle narrowing,” Nat. Methods 11, 579–584 (2014).
[Crossref]

Chizhik, A.

N. Karedla, S. C. Stein, D. Hähnel, I. Gregor, A. Chizhik, and J. Enderlein, “Simultaneous measurement of the three-dimensional orientation of excitation and emission dipoles,” Phys. Rev. Lett. 115, 173002 (2015).
[Crossref]

Chizhik, A. I.

A. I. Chizhik, A. M. Chizhik, A. Huss, R. Jäger, and A. J. Meixner, “Nanoscale probing of dielectric interfaces with single-molecule excitation patterns and radially polarized illumination,” J. Phys. Chem. Lett. 2, 2152–2157 (2011).
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Chizhik, A. M.

A. I. Chizhik, A. M. Chizhik, A. Huss, R. Jäger, and A. J. Meixner, “Nanoscale probing of dielectric interfaces with single-molecule excitation patterns and radially polarized illumination,” J. Phys. Chem. Lett. 2, 2152–2157 (2011).
[Crossref]

Choi, H.

S. Bakshi, H. Choi, N. Rangarajan, K. J. Barns, B. P. Bratton, and J. C. Weisshaar, “Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack,” Appl. Environ. Microbiol. 80, 4977–4986 (2014).
[Crossref]

Chu, S.

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466, 647–651 (2010).
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Churchman, L. S.

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, “Optimized localization analysis for single-molecule tracking and super-resolution microscopy,” Nat. Methods 7, 377–381 (2010).
[Crossref]

Cordek, J. M.

X. Yan, R. C. Habbersett, J. M. Cordek, J. P. Nolan, T. M. Yoshida, J. H. Jett, and B. L. Marrone, “Development of a mechanism-based DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry,” Anal. Biochem. 286, 138–148 (2000).
[Crossref]

Corrie, J. E.

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. Corrie, and Y. E. Goldman, “Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization,” Nature 422, 399–404 (2003).
[Crossref]

Croquette, V.

D. Bensimon, A. J. Simon, V. Croquette, and A. Bensimon, “Stretching DNA with a receding meniscus: experiments and models,” Phys. Rev. Lett. 74, 4754–4757 (1995).
[Crossref]

Cruz, C. A.

C. A. Cruz, H. Shaban Ahmed, A. Kress, N. Bertaux, S. Monneret, M. Mavrakis, J. Savatier, and S. Brasselet, “Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy,” Proc. Natl. Acad. Sci. USA 113, E820–E828 (2016).
[Crossref]

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313, 1642–1645 (2006).
[Crossref]

Dickson, R. M.

R. M. Dickson, D. J. Norris, and W. E. Moerner, “Simultaneous imaging of individual molecules aligned both parallel and perpendicular to the optic axis,” Phys. Rev. Lett. 81, 5322–5325 (1998).
[Crossref]

Douglass, K. M.

L. Carlini, S. J. Holden, K. M. Douglass, and S. Manley, “Correction of a depth-dependent lateral distortion in 3D super-resolution imaging,” Plos One 10, e0142949 (2015).
[Crossref]

Dryden, D.

C. Flors, C. Ravarani, and D. Dryden, “Super-resolution imaging of DNA labeled with intercalating dyes,” ChemPhysChem 10, 2201–2204 (2009).
[Crossref]

Eggeling, C.

Egner, A.

Ehrlich, N.

R. Börner, N. Ehrlich, J. Hohlbein, and C. G. Hübner, “Single molecule 3D orientation in time and space: a 6D dynamic study on fluorescently labeled lipid membranes,” J. Fluoresc., 26, 963–975 (2016).

Ekong, R.

X. Michalet, R. Ekong, F. Fougerousse, S. Rousseaux, C. Schurra, N. Hornigold, M. Slegtenhorst, J. Wolfe, S. Povey, J. S. Beckmann, and A. Bensimon, “Dynamic molecular combing: stretching the whole human genome for high-resolution studies,” Science 277, 1518–1523 (1997).
[Crossref]

Enderle, T.

T. Ha, J. Glass, T. Enderle, D. S. Chemla, and S. Weiss, “Hindered rotational diffusion and rotational jumps of single molecules,” Phys. Rev. Lett. 80, 2093–2096 (1998).
[Crossref]

T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77, 3979–3982 (1996).
[Crossref]

Enderlein, J.

N. Karedla, S. C. Stein, D. Hähnel, I. Gregor, A. Chizhik, and J. Enderlein, “Simultaneous measurement of the three-dimensional orientation of excitation and emission dipoles,” Phys. Rev. Lett. 115, 173002 (2015).
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J. Enderlein, E. Toprak, and P. R. Selvin, “Polarization effect on position accuracy of fluorophore localization,” Opt. Express 14, 8111–8120 (2006).
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E. Toprak, J. Enderlein, S. Syed, S. A. McKinney, R. G. Petschek, T. Ha, Y. E. Goldman, and P. R. Selvin, “Defocused orientation and position imaging (DOPI) of myosin V,” Proc. Natl. Acad. Sci. USA 103, 6495–6499 (2006).
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D. Patra, I. Gregor, and J. Enderlein, “Image analysis of defocused single-molecule images for three-dimensional molecule orientation studies,” J. Phys. Chem. A 108, 6836–6841 (2004).
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M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B 20, 554–559 (2003).
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Engelhardt, J.

J. Engelhardt, J. Keller, P. Hoyer, M. Reuss, T. Staudt, and S. W. Hell, “Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy,” Nano Lett. 11, 209–213 (2011).
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I. Schoen, J. Ries, E. Klotzsch, H. Ewers, and V. Vogel, “Binding-activated localization microscopy of DNA structures,” Nano Lett. 11, 4008–4011 (2011).
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Faraon, A.

M. P. Backlund, A. Arbabi, P. N. Petrov, E. Arbabi, S. Saurabh, A. Faraon, and W. E. Moerner (2016), doi: 10.1038/NPHOTON.2016.93 (to be published).

Fazio, T.

T. Fazio, M. L. Visnapuu, S. Wind, and E. C. Greene, “DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging,” Langmuir 24, 10524–10531 (2008).
[Crossref]

Ferrand, P.

A. Kress, X. Wang, H. Ranchon, J. Savatier, H. Rigneault, P. Ferrand, and S. Brasselet, “Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy,” Biophys. J. 105, 127–136 (2013).
[Crossref]

Flors, C.

C. Flors, C. Ravarani, and D. Dryden, “Super-resolution imaging of DNA labeled with intercalating dyes,” ChemPhysChem 10, 2201–2204 (2009).
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Flyvbjerg, H.

K. I. Mortensen, J. Sung, H. Flyvbjerg, and J. A. Spudich, “Optimized measurements of separations and angles between intra-molecular fluorescent markers,” Nat. Commun. 6, 8621 (2015).
[Crossref]

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, “Optimized localization analysis for single-molecule tracking and super-resolution microscopy,” Nat. Methods 7, 377–381 (2010).
[Crossref]

Forkey, J. N.

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, “Measurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy,” Biophys. J. 89, 1261–1271 (2005).
[Crossref]

M. E. Quinlan, J. N. Forkey, and Y. E. Goldman, “Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy,” Biophys. J. 89, 1132–1142 (2005).
[Crossref]

S. A. Rosenberg, M. E. Quinlan, J. N. Forkey, and Y. E. Goldman, “Rotational motions of macro-molecules by single-molecule fluorescence microscopy,” Acc. Chem. Res. 38, 583–593 (2005).
[Crossref]

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. Corrie, and Y. E. Goldman, “Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization,” Nature 422, 399–404 (2003).
[Crossref]

Fougerousse, F.

X. Michalet, R. Ekong, F. Fougerousse, S. Rousseaux, C. Schurra, N. Hornigold, M. Slegtenhorst, J. Wolfe, S. Povey, J. S. Beckmann, and A. Bensimon, “Dynamic molecular combing: stretching the whole human genome for high-resolution studies,” Science 277, 1518–1523 (1997).
[Crossref]

Fourkas, J. T.

Geisler, C.

Geissbühler, S.

Girirajan, T. P. K.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91, 4258–4272 (2006).
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Gladfelter, A. S.

Glass, J.

T. Ha, J. Glass, T. Enderle, D. S. Chemla, and S. Weiss, “Hindered rotational diffusion and rotational jumps of single molecules,” Phys. Rev. Lett. 80, 2093–2096 (1998).
[Crossref]

Goldman, Y. E.

E. Toprak, J. Enderlein, S. Syed, S. A. McKinney, R. G. Petschek, T. Ha, Y. E. Goldman, and P. R. Selvin, “Defocused orientation and position imaging (DOPI) of myosin V,” Proc. Natl. Acad. Sci. USA 103, 6495–6499 (2006).
[Crossref]

S. A. Rosenberg, M. E. Quinlan, J. N. Forkey, and Y. E. Goldman, “Rotational motions of macro-molecules by single-molecule fluorescence microscopy,” Acc. Chem. Res. 38, 583–593 (2005).
[Crossref]

M. E. Quinlan, J. N. Forkey, and Y. E. Goldman, “Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy,” Biophys. J. 89, 1132–1142 (2005).
[Crossref]

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, “Measurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy,” Biophys. J. 89, 1261–1271 (2005).
[Crossref]

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. Corrie, and Y. E. Goldman, “Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization,” Nature 422, 399–404 (2003).
[Crossref]

Goldner, L. S.

K. D. Weston and L. S. Goldner, “Orientation imaging and reorientation dynamics of single dye molecules,” J. Phys. Chem. B 105, 3453–3462 (2001).
[Crossref]

Goldstein, L. S. B.

H. Sosa, E. J. G. Peterman, W. E. Moerner, and L. S. B. Goldstein, “ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy,” Nat. Struct. Biol. 8, 540–544 (2001).
[Crossref]

E. J. G. Peterman, H. Sosa, L. S. B. Goldstein, and W. E. Moerner, “Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules,” Biophys. J. 81, 2851–2863 (2001).
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Gosse, J. A.

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5, 1027–1030 (2008).
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T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5, 1027–1030 (2008).
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Greene, E. C.

T. Fazio, M. L. Visnapuu, S. Wind, and E. C. Greene, “DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging,” Langmuir 24, 10524–10531 (2008).
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Supplementary Material (1)

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Figures (4)

Fig. 1.
Fig. 1. Experimental overview. (a) By augmenting a conventional fluorescence microscope with an electro-optic modulator (EOM), the excitation laser is modulated to three distinct nearly linear polarization states (pink arrows). The brightness of single molecules, as measured on an electron-multiplying charge coupled device (EMCCD) camera sensor, will vary in proportion to the degree of alignment between the molecule’s absorption dipole moment and the excitation polarization. In principle, we desire a programmable half-wave plate that is normally achieved by aligning the EOM phase modulation axis to 45° with respect to an input linear polarizer (LP) and placing a quarter-wave plate (QWP) after the EOM with the fast axis perpendicular to the linear polarizer. In practice, for different laser lines we used different combinations of dichroics and wave plates to optimize the overall linearity of the rotated polarizations at the sample (see Supplement 1). Inset: The absorption dipole moment is parameterized by an azimuthal angle ϕ and a polar angle θ . Our method permits us to measure the mean azimuthal orientation ϕ of a molecule over the course of a measurement period. In addition, we measure the rotational immobility parameter γ , that quantifies how much a molecule deviates from its mean azimuthal orientation. Using a theoretical model for constrained rotational diffusion, γ is related to an azimuthal arc angle δ , which specifies the full aperture within which the molecule may “wobble.” (b) Our technique is illustrated using fluorescence images of single rhodamine 101 molecules in polymers. Over a series of camera frames, the excitation polarization (denoted by a pink arrow) is set at three different angles and the emission intensity from a single molecule is recorded. ϕ is determined by the relative intensities measured in three consecutive camera frames while γ is related to the overall modulation amplitude of the signal. Images of two relatively immobile molecules (high γ ) embedded in poly(methyl methacrylate) (PMMA) are shown in addition to a rotationally mobile molecule (low γ ) in Mowiol. ϕ values are reported between 0° and 180° due to the inherent two-fold degeneracy of the absorption dipole moment.
Fig. 2.
Fig. 2. Super-resolved images and single-molecule orientation measurements acquired using the intercalating dye SYTOX Orange. (a) Images of λ -DNA strands, color coded according to the mean azimuthal orientation of dye molecules measured within 30 nm voxels. Inset: Due to the intercalative binding mode, absorption dipole moments align perpendicular to the DNA axis. (b) As a DNA strand bends, the mean molecular orientation rotates to remain perpendicular to the DNA axis. This effect is evidenced by the changing color of the curved DNA strands. Inset: A visualization of all orientation measurements localized along a short strip of DNA (green box). The lines point in the direction of ϕ and are color coded to denote γ . (c) A histogram of the orientation measurements localized along the strip of DNA denoted by the white dotted lines in (a). The DNA axis was estimated using spline interpolation. Accordingly, ϕ measurements are reported relative to the DNA axis. The median orientation with respect to the DNA axis is measured to be 87°, and the median absolute deviation is 18°. (d) A histogram of γ measurements corresponding to the same molecules used in (c). δ is estimated to be 44° from the median γ of 0.91. Photon shot noise causes a portion of γ measurements to be greater than 1.
Fig. 3.
Fig. 3. Results acquired using the dye SiR-Hoechst. (a) A super-resolved image of a DNA strand. In this case, the absorption dipole moments of silicon-rhodamine are not constrained because they are not directly bound to DNA strands (right inset). Hence, we elect not to color code our image as no overall alignment with respect to the DNA axis is expected. This feature is evidenced from a visualization of all orientation measurements localized within a small strip of DNA (green boxed region and lower left inset). (b) A histogram of all orientation measurements localized along the strip of DNA denoted by the white dotted lines in (a) (the ϕ measurements are reported relative to the DNA axis). We observe no preferential alignment. (c) A histogram of γ measurements corresponding to the same molecules used in (b). As γ measurements are significantly smaller, the rotational immobility is enhanced and the arc angle δ (estimated from the median γ ) is found to be significantly larger (117°) than that measured for intercalating dyes.
Fig. 4.
Fig. 4. Visualizing bending and tangling of DNA using the intercalator SYTOX Orange. (a) A super-resolution image of a λ -DNA strand containing multiple “bends.” For comparison, a diffraction-limited image was also produced by summing all frames of data contained in our acquisition sequence. (b) Plots of single-molecule positions and orientations demonstrate that when the DNA axis abruptly bends the labeling density drops (see arrows). (c) A super-resolution image of a λ -DNA strand exhibiting “tangles.” (d) By plotting the single-molecule positions and orientations, we show an example of a tangle (arrow) in which dye molecules do not align perpendicular to the DNA axis, as estimated using spline interpolation. The tangle is followed by a region in which few molecules are detected, indicating a possible conformation change in the DNA.

Equations (11)

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U = A T 0 T | μ ( t ) E | 2 d t .
γ = sin ( δ ) δ .
μ = [ cos ( ϕ ) sin ( θ ) sin ( ϕ ) sin ( θ ) cos ( θ ) ] = [ μ x μ y μ z ] .
U = A T E ( 0 T μ ( t ) μ ( t ) d t ) E = A E [ μ x 2 μ x μ y μ x μ z μ x μ y μ y 2 μ y μ z μ x μ z μ y μ z μ z 2 ] E ,
U = A E x y [ μ x 2 μ x μ y μ x μ y μ y 2 ] E x y .
[ U 1 U 2 U 3 ] = A [ | E x , 1 | 2 | E y , 1 | 2 2 R { E x , 1 E y , 1 } | E x , 2 | 2 | E y , 2 | 2 2 R { E x , 2 E y , 2 } | E x , 3 | 2 | E y , 3 | 2 2 R { E x , 3 E y , 3 } ] [ μ x 2 μ y 2 μ x μ y ] = A P [ μ x 2 μ y 2 μ x μ y ] .
A [ μ x 2 μ y 2 μ x μ y ] = P 1 [ U 1 U 2 U 3 ] .
M x y = A [ μ x 2 μ x μ y μ x μ y μ y 2 ] = j = 1 2 λ j v j v j .
M x y = ( λ 1 + λ 2 ) [ γ ( v 1 v 1 ) + ( 1 γ ) I ] .
γ = λ 1 λ 2 λ 1 + λ 2 .
ϕ = a tan 2 ( μ y , μ x ) .

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