Abstract

In fluorescence microscopy, high-speed imaging is often necessary for the proper visualization and analysis of fast subcellular dynamics. Here, we examine how the speed of image acquisition affects the accuracy with which parameters such as the starting position and speed of a microscopic non-stationary fluorescent object can be estimated from the resulting image sequence. Specifically, we use a Fisher information-based performance bound to investigate the detector-dependent effect of frame rate on the accuracy of parameter estimation. We demonstrate that when a charge-coupled device detector is used, the estimation accuracy deteriorates as the frame rate increases beyond a point where the detector’s readout noise begins to overwhelm the low number of photons detected in each frame. In contrast, we show that when an electron-multiplying charge-coupled device (EMCCD) detector is used, the estimation accuracy improves with increasing frame rate. In fact, at high frame rates where the low number of photons detected in each frame renders the fluorescent object difficult to detect visually, imaging with an EMCCD detector represents a natural implementation of the Ultrahigh Accuracy Imaging Modality, and enables estimation with an accuracy approaching that which is attainable only when a hypothetical noiseless detector is used.

© 2014 Optical Society of America

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  1. W. Yang and S. M. Musser, “Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy,” Methods 39(4), 316–328 (2006).
    [CrossRef] [PubMed]
  2. T. Dange, D. Grünwald, A. Grünwald, R. Peters, and U. Kubitscheck, “Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study,” J. Cell Biol. 183(1), 77–86 (2008).
    [CrossRef] [PubMed]
  3. X. Nan, P. A. Sims, P. Chen, and X. S. Xie, “Observation of individual microtubule motor steps in living cells with endocytosed quantum dots,” J. Phys. Chem. B 109(51), 24220–24224 (2005).
    [PubMed]
  4. V. Levi, Q. Ruan, M. Plutz, A. S. Belmont, and E. Gratton, “Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope,” Biophys. J. 89(6), 4275–4285 (2005).
    [CrossRef] [PubMed]
  5. J. Rink, E. Ghigo, Y. Kalaidzidis, and M. Zerial, “Rab conversion as a mechanism of progression from early to late endosomes,” Cell 122(5), 735–749 (2005).
    [CrossRef] [PubMed]
  6. M. P. Taylor, R. Kratchmarov, and L. W. Enquist, “Live cell imaging of alphaherpes virus anterograde transport and spread,” J. Vis. Exp. ( 78), e50723 (2013).
  7. J. Chao, S. Ram, E. S. Ward, and R. J. Ober, “Ultrahigh accuracy imaging modality for super-localization microscopy,” Nat. Methods 10(4), 335–338 (2013).
    [CrossRef] [PubMed]
  8. C. R. Rao, Linear Statistical Inference and its Applications (Wiley, 1965).
  9. S. Ram, E. S. Ward, and R. J. Ober, “A stochastic analysis of performance limits for optical microscopes,” Multidim. Syst. Sign. P. 17(1), 27–57 (2006).
    [CrossRef]
  10. Y. Wong, Z. Lin, and R. J. Ober, “Limit of the accuracy of parameter estimation for moving single molecules imaged by fluorescence microscopy,” IEEE T. Signal Proces. 59(3), 895–911 (2011).
    [CrossRef]
  11. S. M. Kay, Fundamentals of Statistical Signal Processing: Estimation Theory (Prentice Hall PTR, 1993), Vol. I.
  12. J. Chao, E. S. Ward, and R. J. Ober, “Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices,” Multidim. Syst. Sign. P. 23(3), 349–379 (2012).
    [CrossRef]
  13. B. Zhang, J. Zerubia, and J.-C. Olivo-Marin, “Gaussian approximations of fluorescence microscope point-spread function models,” Appl. Optics 46(10), 1819–1829 (2007).
    [CrossRef]
  14. A. Santos and I. T. Young, “Model-based resolution: applying the theory in quantitative microscopy,” Appl. Optics 39(17), 2948–2958 (2000).
    [CrossRef]
  15. M. Born and E. Wolf, Principles of Optics (Cambridge University, 1999).
  16. A. G. Basden, C. A. Haniff, and C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
    [CrossRef]

2013 (2)

M. P. Taylor, R. Kratchmarov, and L. W. Enquist, “Live cell imaging of alphaherpes virus anterograde transport and spread,” J. Vis. Exp. ( 78), e50723 (2013).

J. Chao, S. Ram, E. S. Ward, and R. J. Ober, “Ultrahigh accuracy imaging modality for super-localization microscopy,” Nat. Methods 10(4), 335–338 (2013).
[CrossRef] [PubMed]

2012 (1)

J. Chao, E. S. Ward, and R. J. Ober, “Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices,” Multidim. Syst. Sign. P. 23(3), 349–379 (2012).
[CrossRef]

2011 (1)

Y. Wong, Z. Lin, and R. J. Ober, “Limit of the accuracy of parameter estimation for moving single molecules imaged by fluorescence microscopy,” IEEE T. Signal Proces. 59(3), 895–911 (2011).
[CrossRef]

2008 (1)

T. Dange, D. Grünwald, A. Grünwald, R. Peters, and U. Kubitscheck, “Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study,” J. Cell Biol. 183(1), 77–86 (2008).
[CrossRef] [PubMed]

2007 (1)

B. Zhang, J. Zerubia, and J.-C. Olivo-Marin, “Gaussian approximations of fluorescence microscope point-spread function models,” Appl. Optics 46(10), 1819–1829 (2007).
[CrossRef]

2006 (2)

W. Yang and S. M. Musser, “Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy,” Methods 39(4), 316–328 (2006).
[CrossRef] [PubMed]

S. Ram, E. S. Ward, and R. J. Ober, “A stochastic analysis of performance limits for optical microscopes,” Multidim. Syst. Sign. P. 17(1), 27–57 (2006).
[CrossRef]

2005 (3)

X. Nan, P. A. Sims, P. Chen, and X. S. Xie, “Observation of individual microtubule motor steps in living cells with endocytosed quantum dots,” J. Phys. Chem. B 109(51), 24220–24224 (2005).
[PubMed]

V. Levi, Q. Ruan, M. Plutz, A. S. Belmont, and E. Gratton, “Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope,” Biophys. J. 89(6), 4275–4285 (2005).
[CrossRef] [PubMed]

J. Rink, E. Ghigo, Y. Kalaidzidis, and M. Zerial, “Rab conversion as a mechanism of progression from early to late endosomes,” Cell 122(5), 735–749 (2005).
[CrossRef] [PubMed]

2003 (1)

A. G. Basden, C. A. Haniff, and C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

2000 (1)

A. Santos and I. T. Young, “Model-based resolution: applying the theory in quantitative microscopy,” Appl. Optics 39(17), 2948–2958 (2000).
[CrossRef]

Basden, A. G.

A. G. Basden, C. A. Haniff, and C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

Belmont, A. S.

V. Levi, Q. Ruan, M. Plutz, A. S. Belmont, and E. Gratton, “Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope,” Biophys. J. 89(6), 4275–4285 (2005).
[CrossRef] [PubMed]

Born, M.

M. Born and E. Wolf, Principles of Optics (Cambridge University, 1999).

Chao, J.

J. Chao, S. Ram, E. S. Ward, and R. J. Ober, “Ultrahigh accuracy imaging modality for super-localization microscopy,” Nat. Methods 10(4), 335–338 (2013).
[CrossRef] [PubMed]

J. Chao, E. S. Ward, and R. J. Ober, “Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices,” Multidim. Syst. Sign. P. 23(3), 349–379 (2012).
[CrossRef]

Chen, P.

X. Nan, P. A. Sims, P. Chen, and X. S. Xie, “Observation of individual microtubule motor steps in living cells with endocytosed quantum dots,” J. Phys. Chem. B 109(51), 24220–24224 (2005).
[PubMed]

Dange, T.

T. Dange, D. Grünwald, A. Grünwald, R. Peters, and U. Kubitscheck, “Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study,” J. Cell Biol. 183(1), 77–86 (2008).
[CrossRef] [PubMed]

Enquist, L. W.

M. P. Taylor, R. Kratchmarov, and L. W. Enquist, “Live cell imaging of alphaherpes virus anterograde transport and spread,” J. Vis. Exp. ( 78), e50723 (2013).

Ghigo, E.

J. Rink, E. Ghigo, Y. Kalaidzidis, and M. Zerial, “Rab conversion as a mechanism of progression from early to late endosomes,” Cell 122(5), 735–749 (2005).
[CrossRef] [PubMed]

Gratton, E.

V. Levi, Q. Ruan, M. Plutz, A. S. Belmont, and E. Gratton, “Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope,” Biophys. J. 89(6), 4275–4285 (2005).
[CrossRef] [PubMed]

Grünwald, A.

T. Dange, D. Grünwald, A. Grünwald, R. Peters, and U. Kubitscheck, “Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study,” J. Cell Biol. 183(1), 77–86 (2008).
[CrossRef] [PubMed]

Grünwald, D.

T. Dange, D. Grünwald, A. Grünwald, R. Peters, and U. Kubitscheck, “Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study,” J. Cell Biol. 183(1), 77–86 (2008).
[CrossRef] [PubMed]

Haniff, C. A.

A. G. Basden, C. A. Haniff, and C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

Kalaidzidis, Y.

J. Rink, E. Ghigo, Y. Kalaidzidis, and M. Zerial, “Rab conversion as a mechanism of progression from early to late endosomes,” Cell 122(5), 735–749 (2005).
[CrossRef] [PubMed]

Kay, S. M.

S. M. Kay, Fundamentals of Statistical Signal Processing: Estimation Theory (Prentice Hall PTR, 1993), Vol. I.

Kratchmarov, R.

M. P. Taylor, R. Kratchmarov, and L. W. Enquist, “Live cell imaging of alphaherpes virus anterograde transport and spread,” J. Vis. Exp. ( 78), e50723 (2013).

Kubitscheck, U.

T. Dange, D. Grünwald, A. Grünwald, R. Peters, and U. Kubitscheck, “Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study,” J. Cell Biol. 183(1), 77–86 (2008).
[CrossRef] [PubMed]

Levi, V.

V. Levi, Q. Ruan, M. Plutz, A. S. Belmont, and E. Gratton, “Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope,” Biophys. J. 89(6), 4275–4285 (2005).
[CrossRef] [PubMed]

Lin, Z.

Y. Wong, Z. Lin, and R. J. Ober, “Limit of the accuracy of parameter estimation for moving single molecules imaged by fluorescence microscopy,” IEEE T. Signal Proces. 59(3), 895–911 (2011).
[CrossRef]

Mackay, C. D.

A. G. Basden, C. A. Haniff, and C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

Musser, S. M.

W. Yang and S. M. Musser, “Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy,” Methods 39(4), 316–328 (2006).
[CrossRef] [PubMed]

Nan, X.

X. Nan, P. A. Sims, P. Chen, and X. S. Xie, “Observation of individual microtubule motor steps in living cells with endocytosed quantum dots,” J. Phys. Chem. B 109(51), 24220–24224 (2005).
[PubMed]

Ober, R. J.

J. Chao, S. Ram, E. S. Ward, and R. J. Ober, “Ultrahigh accuracy imaging modality for super-localization microscopy,” Nat. Methods 10(4), 335–338 (2013).
[CrossRef] [PubMed]

J. Chao, E. S. Ward, and R. J. Ober, “Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices,” Multidim. Syst. Sign. P. 23(3), 349–379 (2012).
[CrossRef]

Y. Wong, Z. Lin, and R. J. Ober, “Limit of the accuracy of parameter estimation for moving single molecules imaged by fluorescence microscopy,” IEEE T. Signal Proces. 59(3), 895–911 (2011).
[CrossRef]

S. Ram, E. S. Ward, and R. J. Ober, “A stochastic analysis of performance limits for optical microscopes,” Multidim. Syst. Sign. P. 17(1), 27–57 (2006).
[CrossRef]

Olivo-Marin, J.-C.

B. Zhang, J. Zerubia, and J.-C. Olivo-Marin, “Gaussian approximations of fluorescence microscope point-spread function models,” Appl. Optics 46(10), 1819–1829 (2007).
[CrossRef]

Peters, R.

T. Dange, D. Grünwald, A. Grünwald, R. Peters, and U. Kubitscheck, “Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study,” J. Cell Biol. 183(1), 77–86 (2008).
[CrossRef] [PubMed]

Plutz, M.

V. Levi, Q. Ruan, M. Plutz, A. S. Belmont, and E. Gratton, “Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope,” Biophys. J. 89(6), 4275–4285 (2005).
[CrossRef] [PubMed]

Ram, S.

J. Chao, S. Ram, E. S. Ward, and R. J. Ober, “Ultrahigh accuracy imaging modality for super-localization microscopy,” Nat. Methods 10(4), 335–338 (2013).
[CrossRef] [PubMed]

S. Ram, E. S. Ward, and R. J. Ober, “A stochastic analysis of performance limits for optical microscopes,” Multidim. Syst. Sign. P. 17(1), 27–57 (2006).
[CrossRef]

Rao, C. R.

C. R. Rao, Linear Statistical Inference and its Applications (Wiley, 1965).

Rink, J.

J. Rink, E. Ghigo, Y. Kalaidzidis, and M. Zerial, “Rab conversion as a mechanism of progression from early to late endosomes,” Cell 122(5), 735–749 (2005).
[CrossRef] [PubMed]

Ruan, Q.

V. Levi, Q. Ruan, M. Plutz, A. S. Belmont, and E. Gratton, “Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope,” Biophys. J. 89(6), 4275–4285 (2005).
[CrossRef] [PubMed]

Santos, A.

A. Santos and I. T. Young, “Model-based resolution: applying the theory in quantitative microscopy,” Appl. Optics 39(17), 2948–2958 (2000).
[CrossRef]

Sims, P. A.

X. Nan, P. A. Sims, P. Chen, and X. S. Xie, “Observation of individual microtubule motor steps in living cells with endocytosed quantum dots,” J. Phys. Chem. B 109(51), 24220–24224 (2005).
[PubMed]

Taylor, M. P.

M. P. Taylor, R. Kratchmarov, and L. W. Enquist, “Live cell imaging of alphaherpes virus anterograde transport and spread,” J. Vis. Exp. ( 78), e50723 (2013).

Ward, E. S.

J. Chao, S. Ram, E. S. Ward, and R. J. Ober, “Ultrahigh accuracy imaging modality for super-localization microscopy,” Nat. Methods 10(4), 335–338 (2013).
[CrossRef] [PubMed]

J. Chao, E. S. Ward, and R. J. Ober, “Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices,” Multidim. Syst. Sign. P. 23(3), 349–379 (2012).
[CrossRef]

S. Ram, E. S. Ward, and R. J. Ober, “A stochastic analysis of performance limits for optical microscopes,” Multidim. Syst. Sign. P. 17(1), 27–57 (2006).
[CrossRef]

Wolf, E.

M. Born and E. Wolf, Principles of Optics (Cambridge University, 1999).

Wong, Y.

Y. Wong, Z. Lin, and R. J. Ober, “Limit of the accuracy of parameter estimation for moving single molecules imaged by fluorescence microscopy,” IEEE T. Signal Proces. 59(3), 895–911 (2011).
[CrossRef]

Xie, X. S.

X. Nan, P. A. Sims, P. Chen, and X. S. Xie, “Observation of individual microtubule motor steps in living cells with endocytosed quantum dots,” J. Phys. Chem. B 109(51), 24220–24224 (2005).
[PubMed]

Yang, W.

W. Yang and S. M. Musser, “Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy,” Methods 39(4), 316–328 (2006).
[CrossRef] [PubMed]

Young, I. T.

A. Santos and I. T. Young, “Model-based resolution: applying the theory in quantitative microscopy,” Appl. Optics 39(17), 2948–2958 (2000).
[CrossRef]

Zerial, M.

J. Rink, E. Ghigo, Y. Kalaidzidis, and M. Zerial, “Rab conversion as a mechanism of progression from early to late endosomes,” Cell 122(5), 735–749 (2005).
[CrossRef] [PubMed]

Zerubia, J.

B. Zhang, J. Zerubia, and J.-C. Olivo-Marin, “Gaussian approximations of fluorescence microscope point-spread function models,” Appl. Optics 46(10), 1819–1829 (2007).
[CrossRef]

Zhang, B.

B. Zhang, J. Zerubia, and J.-C. Olivo-Marin, “Gaussian approximations of fluorescence microscope point-spread function models,” Appl. Optics 46(10), 1819–1829 (2007).
[CrossRef]

Appl. Optics (2)

B. Zhang, J. Zerubia, and J.-C. Olivo-Marin, “Gaussian approximations of fluorescence microscope point-spread function models,” Appl. Optics 46(10), 1819–1829 (2007).
[CrossRef]

A. Santos and I. T. Young, “Model-based resolution: applying the theory in quantitative microscopy,” Appl. Optics 39(17), 2948–2958 (2000).
[CrossRef]

Biophys. J. (1)

V. Levi, Q. Ruan, M. Plutz, A. S. Belmont, and E. Gratton, “Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope,” Biophys. J. 89(6), 4275–4285 (2005).
[CrossRef] [PubMed]

Cell (1)

J. Rink, E. Ghigo, Y. Kalaidzidis, and M. Zerial, “Rab conversion as a mechanism of progression from early to late endosomes,” Cell 122(5), 735–749 (2005).
[CrossRef] [PubMed]

IEEE T. Signal Proces. (1)

Y. Wong, Z. Lin, and R. J. Ober, “Limit of the accuracy of parameter estimation for moving single molecules imaged by fluorescence microscopy,” IEEE T. Signal Proces. 59(3), 895–911 (2011).
[CrossRef]

J. Cell Biol. (1)

T. Dange, D. Grünwald, A. Grünwald, R. Peters, and U. Kubitscheck, “Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study,” J. Cell Biol. 183(1), 77–86 (2008).
[CrossRef] [PubMed]

J. Phys. Chem. B (1)

X. Nan, P. A. Sims, P. Chen, and X. S. Xie, “Observation of individual microtubule motor steps in living cells with endocytosed quantum dots,” J. Phys. Chem. B 109(51), 24220–24224 (2005).
[PubMed]

J. Vis. Exp. (1)

M. P. Taylor, R. Kratchmarov, and L. W. Enquist, “Live cell imaging of alphaherpes virus anterograde transport and spread,” J. Vis. Exp. ( 78), e50723 (2013).

Methods (1)

W. Yang and S. M. Musser, “Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy,” Methods 39(4), 316–328 (2006).
[CrossRef] [PubMed]

Mon. Not. R. Astron. Soc. (1)

A. G. Basden, C. A. Haniff, and C. D. Mackay, “Photon counting strategies with low-light-level CCDs,” Mon. Not. R. Astron. Soc. 345(3), 985–991 (2003).
[CrossRef]

Multidim. Syst. Sign. P. (2)

J. Chao, E. S. Ward, and R. J. Ober, “Fisher information matrix for branching processes with application to electron-multiplying charge-coupled devices,” Multidim. Syst. Sign. P. 23(3), 349–379 (2012).
[CrossRef]

S. Ram, E. S. Ward, and R. J. Ober, “A stochastic analysis of performance limits for optical microscopes,” Multidim. Syst. Sign. P. 17(1), 27–57 (2006).
[CrossRef]

Nat. Methods (1)

J. Chao, S. Ram, E. S. Ward, and R. J. Ober, “Ultrahigh accuracy imaging modality for super-localization microscopy,” Nat. Methods 10(4), 335–338 (2013).
[CrossRef] [PubMed]

Other (3)

C. R. Rao, Linear Statistical Inference and its Applications (Wiley, 1965).

S. M. Kay, Fundamentals of Statistical Signal Processing: Estimation Theory (Prentice Hall PTR, 1993), Vol. I.

M. Born and E. Wolf, Principles of Optics (Cambridge University, 1999).

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