Abstract

The electron-multiplying charge-coupled device (EMCCD) camera possesses an electron multiplying function that can effectively convert the weak incident photon signal to amplified electron output, thereby greatly enhancing the contrast of the acquired images. This device has become a popular photon detector in single-cell biophysical assays to enhance subcellular images. However, the quantitative relationship between the resolution in such measurements and the electron multiplication setting in the EMCCD camera is not well-understood. We therefore developed a method to characterize the exact dependence of the signal-to-noise-ratio (SNR) on EM gain settings over a full range of incident light intensity. This information was further used to evaluate the EMCCD performance in subcellular particle tracking. We conclude that there are optimal EM gain settings for achieving the best SNR and the best spatial resolution in these experiments. If it is not used optimally, electron multiplication can decrease the SNR and increases spatial error.

© 2010 OSA

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    [CrossRef] [PubMed]

2009

G. Fink, L. Hajdo, K. J. Skowronek, C. Reuther, A. A. Kasprzak, and S. Diez, “The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding,” Nat. Cell Biol. 11(6), 717–723 (2009).
[CrossRef] [PubMed]

L. Zhang, L. Neves, J. S. Lundeen, and I. A. Walmsley, “A Characterization of the Single-photon Sensitivity of an Electron Multiplying Charge-Coupled Device,” J. Phys. B 42(11), 114011 (2009).
[CrossRef]

P. H. Wu, S. H. Arce, P. R. Burney, and Y. Tseng, “A novel approach to high accuracy of video-based microrheology,” Biophys. J. 96(12), 5103–5111 (2009).
[CrossRef] [PubMed]

2008

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

S. Li, S. L. Lian, J. J. Moser, M. L. Fritzler, M. J. Fritzler, M. Satoh, and E. K. Chan, “Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing,” J. Cell Sci. 121(24), 4134–4144 (2008).
[CrossRef] [PubMed]

M. Jonas, H. Huang, R. D. Kamm, and P. T. So, “Fast fluorescence laser tracking microrheometry. I: instrument development,” Biophys. J. 94(4), 1459–1469 (2008).
[CrossRef]

J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[CrossRef] [PubMed]

A. A. Gerencser, J. Doczi, B. Töröcsik, E. Bossy-Wetzel, and V. Adam-Vizi, “Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte-neuron differences,” Biophys. J. 95(5), 2583–2598 (2008).
[CrossRef] [PubMed]

2007

E. S. Levitan, F. Lanni, and D. Shakiryanova, “In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction,” Nat. Protoc. 2(5), 1117–1125 (2007).
[CrossRef] [PubMed]

C. Bakal, J. Aach, G. Church, and N. Perrimon, “Quantitative morphological signatures define local signaling networks regulating cell morphology,” Science 316(5832), 1753–1756 (2007).
[CrossRef] [PubMed]

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

2005

T. Savin and P. S. Doyle, “Static and dynamic errors in particle tracking microrheology,” Biophys. J. 88(1), 623–638 (2005).
[CrossRef]

2004

S. M. Görisch, M. Wachsmuth, C. Ittrich, C. P. Bacher, K. Rippe, and P. Lichter, “Nuclear body movement is determined by chromatin accessibility and dynamics,” Proc. Natl. Acad. Sci. U.S.A. 101(36), 13221–13226 (2004).
[CrossRef] [PubMed]

T. P. Kole, Y. Tseng, L. Huang, J. L. Katz, and D. Wirtz, “Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation,” Mol. Biol. Cell 15(7), 3475–3484 (2004).
[CrossRef] [PubMed]

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303(5658), 676–678 (2004).
[CrossRef]

2003

M. S. Robbins and B. J. Hadwen, “The noise performance of electron multiplying charge-coupled devices,” IEEE Trans. Electron. Dev. 50(5), 1227–1232 (2003).
[CrossRef]

C. H. Eskiw, G. Dellaire, J. S. Mymryk, and D. P. Bazett-Jones, “Size, position and dynamic behavior of PML nuclear bodies following cell stress as a paradigm for supramolecular trafficking and assembly,” J. Cell Sci. 116(21), 4455–4466 (2003).
[CrossRef] [PubMed]

Y. Reibel, M. Jung, M. Bouhifd, B. Cunin, and C. Draman, “CCD or CMOS Camera Noise Characterisation,” Eur. Phys. J. D 21, 75–80 (2003).

2002

J. Y. Xu, Y. Tseng, C. J. Carriere, and D. Wirtz, “Microheterogeneity and microrheology of wheat gliadin suspensions studied by multiple-particle tracking,” Biomacromolecules 3(1), 92–99 (2002).
[CrossRef] [PubMed]

2001

J. Hynecek, “Impactron-a new solid state image intensifier,” IEEE Trans. Electron. Dev. 48(10), 2238–2241 (2001).
[CrossRef]

2000

M. Benoit, D. Gabriel, G. Gerisch, and H. E. Gaub, “Discrete interactions in cell adhesion measured by single-molecule force spectroscopy,” Nat. Cell Biol. 2(6), 313–317 (2000).
[CrossRef] [PubMed]

1999

Y. Chen, J. D. Müller, P. T. So, and E. Gratton, “The photon counting histogram in fluorescence fluctuation spectroscopy,” Biophys. J. 77(1), 553–567 (1999).
[CrossRef] [PubMed]

1994

J. C. Mullikin, L. J. van Vliet, H. Netten, F. R. Boddeke, G. van der Feltz, and I. T. Young, “Methods For CCD Camera Characterization,” SPIE Image Acquis. Sci. Imaging Syst. 2173, 73–84 (1994).

Aach, J.

C. Bakal, J. Aach, G. Church, and N. Perrimon, “Quantitative morphological signatures define local signaling networks regulating cell morphology,” Science 316(5832), 1753–1756 (2007).
[CrossRef] [PubMed]

Adam-Vizi, V.

A. A. Gerencser, J. Doczi, B. Töröcsik, E. Bossy-Wetzel, and V. Adam-Vizi, “Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte-neuron differences,” Biophys. J. 95(5), 2583–2598 (2008).
[CrossRef] [PubMed]

Ahmed, S.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Arce, S. H.

P. H. Wu, S. H. Arce, P. R. Burney, and Y. Tseng, “A novel approach to high accuracy of video-based microrheology,” Biophys. J. 96(12), 5103–5111 (2009).
[CrossRef] [PubMed]

Bacher, C. P.

S. M. Görisch, M. Wachsmuth, C. Ittrich, C. P. Bacher, K. Rippe, and P. Lichter, “Nuclear body movement is determined by chromatin accessibility and dynamics,” Proc. Natl. Acad. Sci. U.S.A. 101(36), 13221–13226 (2004).
[CrossRef] [PubMed]

Bakal, C.

C. Bakal, J. Aach, G. Church, and N. Perrimon, “Quantitative morphological signatures define local signaling networks regulating cell morphology,” Science 316(5832), 1753–1756 (2007).
[CrossRef] [PubMed]

Bazett-Jones, D. P.

C. H. Eskiw, G. Dellaire, J. S. Mymryk, and D. P. Bazett-Jones, “Size, position and dynamic behavior of PML nuclear bodies following cell stress as a paradigm for supramolecular trafficking and assembly,” J. Cell Sci. 116(21), 4455–4466 (2003).
[CrossRef] [PubMed]

Benoit, M.

M. Benoit, D. Gabriel, G. Gerisch, and H. E. Gaub, “Discrete interactions in cell adhesion measured by single-molecule force spectroscopy,” Nat. Cell Biol. 2(6), 313–317 (2000).
[CrossRef] [PubMed]

Boddeke, F. R.

J. C. Mullikin, L. J. van Vliet, H. Netten, F. R. Boddeke, G. van der Feltz, and I. T. Young, “Methods For CCD Camera Characterization,” SPIE Image Acquis. Sci. Imaging Syst. 2173, 73–84 (1994).

Bossy-Wetzel, E.

A. A. Gerencser, J. Doczi, B. Töröcsik, E. Bossy-Wetzel, and V. Adam-Vizi, “Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte-neuron differences,” Biophys. J. 95(5), 2583–2598 (2008).
[CrossRef] [PubMed]

Bouhifd, M.

Y. Reibel, M. Jung, M. Bouhifd, B. Cunin, and C. Draman, “CCD or CMOS Camera Noise Characterisation,” Eur. Phys. J. D 21, 75–80 (2003).

Burney, P. R.

P. H. Wu, S. H. Arce, P. R. Burney, and Y. Tseng, “A novel approach to high accuracy of video-based microrheology,” Biophys. J. 96(12), 5103–5111 (2009).
[CrossRef] [PubMed]

Carriere, C. J.

J. Y. Xu, Y. Tseng, C. J. Carriere, and D. Wirtz, “Microheterogeneity and microrheology of wheat gliadin suspensions studied by multiple-particle tracking,” Biomacromolecules 3(1), 92–99 (2002).
[CrossRef] [PubMed]

Chan, E. K.

S. Li, S. L. Lian, J. J. Moser, M. L. Fritzler, M. J. Fritzler, M. Satoh, and E. K. Chan, “Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing,” J. Cell Sci. 121(24), 4134–4144 (2008).
[CrossRef] [PubMed]

Chen, Y.

Y. Chen, J. D. Müller, P. T. So, and E. Gratton, “The photon counting histogram in fluorescence fluctuation spectroscopy,” Biophys. J. 77(1), 553–567 (1999).
[CrossRef] [PubMed]

Church, G.

C. Bakal, J. Aach, G. Church, and N. Perrimon, “Quantitative morphological signatures define local signaling networks regulating cell morphology,” Science 316(5832), 1753–1756 (2007).
[CrossRef] [PubMed]

Cunin, B.

Y. Reibel, M. Jung, M. Bouhifd, B. Cunin, and C. Draman, “CCD or CMOS Camera Noise Characterisation,” Eur. Phys. J. D 21, 75–80 (2003).

Dellaire, G.

C. H. Eskiw, G. Dellaire, J. S. Mymryk, and D. P. Bazett-Jones, “Size, position and dynamic behavior of PML nuclear bodies following cell stress as a paradigm for supramolecular trafficking and assembly,” J. Cell Sci. 116(21), 4455–4466 (2003).
[CrossRef] [PubMed]

Diez, S.

G. Fink, L. Hajdo, K. J. Skowronek, C. Reuther, A. A. Kasprzak, and S. Diez, “The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding,” Nat. Cell Biol. 11(6), 717–723 (2009).
[CrossRef] [PubMed]

Dittrich, P.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Doczi, J.

A. A. Gerencser, J. Doczi, B. Töröcsik, E. Bossy-Wetzel, and V. Adam-Vizi, “Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte-neuron differences,” Biophys. J. 95(5), 2583–2598 (2008).
[CrossRef] [PubMed]

Doyle, P. S.

T. Savin and P. S. Doyle, “Static and dynamic errors in particle tracking microrheology,” Biophys. J. 88(1), 623–638 (2005).
[CrossRef]

Draman, C.

Y. Reibel, M. Jung, M. Bouhifd, B. Cunin, and C. Draman, “CCD or CMOS Camera Noise Characterisation,” Eur. Phys. J. D 21, 75–80 (2003).

Eskiw, C. H.

C. H. Eskiw, G. Dellaire, J. S. Mymryk, and D. P. Bazett-Jones, “Size, position and dynamic behavior of PML nuclear bodies following cell stress as a paradigm for supramolecular trafficking and assembly,” J. Cell Sci. 116(21), 4455–4466 (2003).
[CrossRef] [PubMed]

Fink, G.

G. Fink, L. Hajdo, K. J. Skowronek, C. Reuther, A. A. Kasprzak, and S. Diez, “The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding,” Nat. Cell Biol. 11(6), 717–723 (2009).
[CrossRef] [PubMed]

Fritzler, M. J.

S. Li, S. L. Lian, J. J. Moser, M. L. Fritzler, M. J. Fritzler, M. Satoh, and E. K. Chan, “Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing,” J. Cell Sci. 121(24), 4134–4144 (2008).
[CrossRef] [PubMed]

Fritzler, M. L.

S. Li, S. L. Lian, J. J. Moser, M. L. Fritzler, M. J. Fritzler, M. Satoh, and E. K. Chan, “Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing,” J. Cell Sci. 121(24), 4134–4144 (2008).
[CrossRef] [PubMed]

Gabriel, D.

M. Benoit, D. Gabriel, G. Gerisch, and H. E. Gaub, “Discrete interactions in cell adhesion measured by single-molecule force spectroscopy,” Nat. Cell Biol. 2(6), 313–317 (2000).
[CrossRef] [PubMed]

Gaub, H. E.

M. Benoit, D. Gabriel, G. Gerisch, and H. E. Gaub, “Discrete interactions in cell adhesion measured by single-molecule force spectroscopy,” Nat. Cell Biol. 2(6), 313–317 (2000).
[CrossRef] [PubMed]

Gerencser, A. A.

A. A. Gerencser, J. Doczi, B. Töröcsik, E. Bossy-Wetzel, and V. Adam-Vizi, “Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte-neuron differences,” Biophys. J. 95(5), 2583–2598 (2008).
[CrossRef] [PubMed]

Gerisch, G.

M. Benoit, D. Gabriel, G. Gerisch, and H. E. Gaub, “Discrete interactions in cell adhesion measured by single-molecule force spectroscopy,” Nat. Cell Biol. 2(6), 313–317 (2000).
[CrossRef] [PubMed]

Gerstner, N.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Görisch, S. M.

S. M. Görisch, M. Wachsmuth, C. Ittrich, C. P. Bacher, K. Rippe, and P. Lichter, “Nuclear body movement is determined by chromatin accessibility and dynamics,” Proc. Natl. Acad. Sci. U.S.A. 101(36), 13221–13226 (2004).
[CrossRef] [PubMed]

Gratton, E.

J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[CrossRef] [PubMed]

Y. Chen, J. D. Müller, P. T. So, and E. Gratton, “The photon counting histogram in fluorescence fluctuation spectroscopy,” Biophys. J. 77(1), 553–567 (1999).
[CrossRef] [PubMed]

Guo, L.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Hadwen, B. J.

M. S. Robbins and B. J. Hadwen, “The noise performance of electron multiplying charge-coupled devices,” IEEE Trans. Electron. Dev. 50(5), 1227–1232 (2003).
[CrossRef]

Hajdo, L.

G. Fink, L. Hajdo, K. J. Skowronek, C. Reuther, A. A. Kasprzak, and S. Diez, “The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding,” Nat. Cell Biol. 11(6), 717–723 (2009).
[CrossRef] [PubMed]

Hemmerich, P.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Hofmann, T. G.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Hoischen, C.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Huang, H.

M. Jonas, H. Huang, R. D. Kamm, and P. T. So, “Fast fluorescence laser tracking microrheometry. I: instrument development,” Biophys. J. 94(4), 1459–1469 (2008).
[CrossRef]

Huang, L.

T. P. Kole, Y. Tseng, L. Huang, J. L. Katz, and D. Wirtz, “Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation,” Mol. Biol. Cell 15(7), 3475–3484 (2004).
[CrossRef] [PubMed]

Hynecek, J.

J. Hynecek, “Impactron-a new solid state image intensifier,” IEEE Trans. Electron. Dev. 48(10), 2238–2241 (2001).
[CrossRef]

Ittrich, C.

S. M. Görisch, M. Wachsmuth, C. Ittrich, C. P. Bacher, K. Rippe, and P. Lichter, “Nuclear body movement is determined by chromatin accessibility and dynamics,” Proc. Natl. Acad. Sci. U.S.A. 101(36), 13221–13226 (2004).
[CrossRef] [PubMed]

Jonas, M.

M. Jonas, H. Huang, R. D. Kamm, and P. T. So, “Fast fluorescence laser tracking microrheometry. I: instrument development,” Biophys. J. 94(4), 1459–1469 (2008).
[CrossRef]

Jung, M.

Y. Reibel, M. Jung, M. Bouhifd, B. Cunin, and C. Draman, “CCD or CMOS Camera Noise Characterisation,” Eur. Phys. J. D 21, 75–80 (2003).

Kamm, R. D.

M. Jonas, H. Huang, R. D. Kamm, and P. T. So, “Fast fluorescence laser tracking microrheometry. I: instrument development,” Biophys. J. 94(4), 1459–1469 (2008).
[CrossRef]

Kannan, B.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Kasprzak, A. A.

G. Fink, L. Hajdo, K. J. Skowronek, C. Reuther, A. A. Kasprzak, and S. Diez, “The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding,” Nat. Cell Biol. 11(6), 717–723 (2009).
[CrossRef] [PubMed]

Katz, J. L.

T. P. Kole, Y. Tseng, L. Huang, J. L. Katz, and D. Wirtz, “Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation,” Mol. Biol. Cell 15(7), 3475–3484 (2004).
[CrossRef] [PubMed]

Kole, T. P.

T. P. Kole, Y. Tseng, L. Huang, J. L. Katz, and D. Wirtz, “Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation,” Mol. Biol. Cell 15(7), 3475–3484 (2004).
[CrossRef] [PubMed]

Lanni, F.

E. S. Levitan, F. Lanni, and D. Shakiryanova, “In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction,” Nat. Protoc. 2(5), 1117–1125 (2007).
[CrossRef] [PubMed]

Lenser, T.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Levitan, E. S.

E. S. Levitan, F. Lanni, and D. Shakiryanova, “In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction,” Nat. Protoc. 2(5), 1117–1125 (2007).
[CrossRef] [PubMed]

Li, S.

S. Li, S. L. Lian, J. J. Moser, M. L. Fritzler, M. J. Fritzler, M. Satoh, and E. K. Chan, “Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing,” J. Cell Sci. 121(24), 4134–4144 (2008).
[CrossRef] [PubMed]

Lian, S. L.

S. Li, S. L. Lian, J. J. Moser, M. L. Fritzler, M. J. Fritzler, M. Satoh, and E. K. Chan, “Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing,” J. Cell Sci. 121(24), 4134–4144 (2008).
[CrossRef] [PubMed]

Lichter, P.

S. M. Görisch, M. Wachsmuth, C. Ittrich, C. P. Bacher, K. Rippe, and P. Lichter, “Nuclear body movement is determined by chromatin accessibility and dynamics,” Proc. Natl. Acad. Sci. U.S.A. 101(36), 13221–13226 (2004).
[CrossRef] [PubMed]

Lundeen, J. S.

L. Zhang, L. Neves, J. S. Lundeen, and I. A. Walmsley, “A Characterization of the Single-photon Sensitivity of an Electron Multiplying Charge-Coupled Device,” J. Phys. B 42(11), 114011 (2009).
[CrossRef]

Maruyama, I.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Maul, G.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Moser, J. J.

S. Li, S. L. Lian, J. J. Moser, M. L. Fritzler, M. J. Fritzler, M. Satoh, and E. K. Chan, “Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing,” J. Cell Sci. 121(24), 4134–4144 (2008).
[CrossRef] [PubMed]

Müller, J. D.

Y. Chen, J. D. Müller, P. T. So, and E. Gratton, “The photon counting histogram in fluorescence fluctuation spectroscopy,” Biophys. J. 77(1), 553–567 (1999).
[CrossRef] [PubMed]

Mullikin, J. C.

J. C. Mullikin, L. J. van Vliet, H. Netten, F. R. Boddeke, G. van der Feltz, and I. T. Young, “Methods For CCD Camera Characterization,” SPIE Image Acquis. Sci. Imaging Syst. 2173, 73–84 (1994).

Mymryk, J. S.

C. H. Eskiw, G. Dellaire, J. S. Mymryk, and D. P. Bazett-Jones, “Size, position and dynamic behavior of PML nuclear bodies following cell stress as a paradigm for supramolecular trafficking and assembly,” J. Cell Sci. 116(21), 4455–4466 (2003).
[CrossRef] [PubMed]

Negorev, D.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Netten, H.

J. C. Mullikin, L. J. van Vliet, H. Netten, F. R. Boddeke, G. van der Feltz, and I. T. Young, “Methods For CCD Camera Characterization,” SPIE Image Acquis. Sci. Imaging Syst. 2173, 73–84 (1994).

Neves, L.

L. Zhang, L. Neves, J. S. Lundeen, and I. A. Walmsley, “A Characterization of the Single-photon Sensitivity of an Electron Multiplying Charge-Coupled Device,” J. Phys. B 42(11), 114011 (2009).
[CrossRef]

Perrimon, N.

C. Bakal, J. Aach, G. Church, and N. Perrimon, “Quantitative morphological signatures define local signaling networks regulating cell morphology,” Science 316(5832), 1753–1756 (2007).
[CrossRef] [PubMed]

Reibel, Y.

Y. Reibel, M. Jung, M. Bouhifd, B. Cunin, and C. Draman, “CCD or CMOS Camera Noise Characterisation,” Eur. Phys. J. D 21, 75–80 (2003).

Reuther, C.

G. Fink, L. Hajdo, K. J. Skowronek, C. Reuther, A. A. Kasprzak, and S. Diez, “The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding,” Nat. Cell Biol. 11(6), 717–723 (2009).
[CrossRef] [PubMed]

Rippe, K.

S. M. Görisch, M. Wachsmuth, C. Ittrich, C. P. Bacher, K. Rippe, and P. Lichter, “Nuclear body movement is determined by chromatin accessibility and dynamics,” Proc. Natl. Acad. Sci. U.S.A. 101(36), 13221–13226 (2004).
[CrossRef] [PubMed]

Robbins, M. S.

M. S. Robbins and B. J. Hadwen, “The noise performance of electron multiplying charge-coupled devices,” IEEE Trans. Electron. Dev. 50(5), 1227–1232 (2003).
[CrossRef]

Satoh, M.

S. Li, S. L. Lian, J. J. Moser, M. L. Fritzler, M. J. Fritzler, M. Satoh, and E. K. Chan, “Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing,” J. Cell Sci. 121(24), 4134–4144 (2008).
[CrossRef] [PubMed]

Savin, T.

T. Savin and P. S. Doyle, “Static and dynamic errors in particle tracking microrheology,” Biophys. J. 88(1), 623–638 (2005).
[CrossRef]

Schwanitz, G.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Selvin, P. R.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303(5658), 676–678 (2004).
[CrossRef]

Shakiryanova, D.

E. S. Levitan, F. Lanni, and D. Shakiryanova, “In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction,” Nat. Protoc. 2(5), 1117–1125 (2007).
[CrossRef] [PubMed]

Skowronek, K. J.

G. Fink, L. Hajdo, K. J. Skowronek, C. Reuther, A. A. Kasprzak, and S. Diez, “The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding,” Nat. Cell Biol. 11(6), 717–723 (2009).
[CrossRef] [PubMed]

So, P. T.

M. Jonas, H. Huang, R. D. Kamm, and P. T. So, “Fast fluorescence laser tracking microrheometry. I: instrument development,” Biophys. J. 94(4), 1459–1469 (2008).
[CrossRef]

Y. Chen, J. D. Müller, P. T. So, and E. Gratton, “The photon counting histogram in fluorescence fluctuation spectroscopy,” Biophys. J. 77(1), 553–567 (1999).
[CrossRef] [PubMed]

Sudhaharan, T.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Tomishige, M.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303(5658), 676–678 (2004).
[CrossRef]

Töröcsik, B.

A. A. Gerencser, J. Doczi, B. Töröcsik, E. Bossy-Wetzel, and V. Adam-Vizi, “Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte-neuron differences,” Biophys. J. 95(5), 2583–2598 (2008).
[CrossRef] [PubMed]

Tseng, Y.

P. H. Wu, S. H. Arce, P. R. Burney, and Y. Tseng, “A novel approach to high accuracy of video-based microrheology,” Biophys. J. 96(12), 5103–5111 (2009).
[CrossRef] [PubMed]

T. P. Kole, Y. Tseng, L. Huang, J. L. Katz, and D. Wirtz, “Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation,” Mol. Biol. Cell 15(7), 3475–3484 (2004).
[CrossRef] [PubMed]

J. Y. Xu, Y. Tseng, C. J. Carriere, and D. Wirtz, “Microheterogeneity and microrheology of wheat gliadin suspensions studied by multiple-particle tracking,” Biomacromolecules 3(1), 92–99 (2002).
[CrossRef] [PubMed]

Unruh, J. R.

J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[CrossRef] [PubMed]

Vale, R. D.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303(5658), 676–678 (2004).
[CrossRef]

van der Feltz, G.

J. C. Mullikin, L. J. van Vliet, H. Netten, F. R. Boddeke, G. van der Feltz, and I. T. Young, “Methods For CCD Camera Characterization,” SPIE Image Acquis. Sci. Imaging Syst. 2173, 73–84 (1994).

van Vliet, L. J.

J. C. Mullikin, L. J. van Vliet, H. Netten, F. R. Boddeke, G. van der Feltz, and I. T. Young, “Methods For CCD Camera Characterization,” SPIE Image Acquis. Sci. Imaging Syst. 2173, 73–84 (1994).

Wachsmuth, M.

S. M. Görisch, M. Wachsmuth, C. Ittrich, C. P. Bacher, K. Rippe, and P. Lichter, “Nuclear body movement is determined by chromatin accessibility and dynamics,” Proc. Natl. Acad. Sci. U.S.A. 101(36), 13221–13226 (2004).
[CrossRef] [PubMed]

Walmsley, I. A.

L. Zhang, L. Neves, J. S. Lundeen, and I. A. Walmsley, “A Characterization of the Single-photon Sensitivity of an Electron Multiplying Charge-Coupled Device,” J. Phys. B 42(11), 114011 (2009).
[CrossRef]

Weidtkamp-Peters, S.

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

Wirtz, D.

T. P. Kole, Y. Tseng, L. Huang, J. L. Katz, and D. Wirtz, “Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation,” Mol. Biol. Cell 15(7), 3475–3484 (2004).
[CrossRef] [PubMed]

J. Y. Xu, Y. Tseng, C. J. Carriere, and D. Wirtz, “Microheterogeneity and microrheology of wheat gliadin suspensions studied by multiple-particle tracking,” Biomacromolecules 3(1), 92–99 (2002).
[CrossRef] [PubMed]

Wohland, T.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Wu, P. H.

P. H. Wu, S. H. Arce, P. R. Burney, and Y. Tseng, “A novel approach to high accuracy of video-based microrheology,” Biophys. J. 96(12), 5103–5111 (2009).
[CrossRef] [PubMed]

Xu, J. Y.

J. Y. Xu, Y. Tseng, C. J. Carriere, and D. Wirtz, “Microheterogeneity and microrheology of wheat gliadin suspensions studied by multiple-particle tracking,” Biomacromolecules 3(1), 92–99 (2002).
[CrossRef] [PubMed]

Yildiz, A.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303(5658), 676–678 (2004).
[CrossRef]

Young, I. T.

J. C. Mullikin, L. J. van Vliet, H. Netten, F. R. Boddeke, G. van der Feltz, and I. T. Young, “Methods For CCD Camera Characterization,” SPIE Image Acquis. Sci. Imaging Syst. 2173, 73–84 (1994).

Zhang, L.

L. Zhang, L. Neves, J. S. Lundeen, and I. A. Walmsley, “A Characterization of the Single-photon Sensitivity of an Electron Multiplying Charge-Coupled Device,” J. Phys. B 42(11), 114011 (2009).
[CrossRef]

Anal. Chem.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Biomacromolecules

J. Y. Xu, Y. Tseng, C. J. Carriere, and D. Wirtz, “Microheterogeneity and microrheology of wheat gliadin suspensions studied by multiple-particle tracking,” Biomacromolecules 3(1), 92–99 (2002).
[CrossRef] [PubMed]

Biophys. J.

M. Jonas, H. Huang, R. D. Kamm, and P. T. So, “Fast fluorescence laser tracking microrheometry. I: instrument development,” Biophys. J. 94(4), 1459–1469 (2008).
[CrossRef]

Y. Chen, J. D. Müller, P. T. So, and E. Gratton, “The photon counting histogram in fluorescence fluctuation spectroscopy,” Biophys. J. 77(1), 553–567 (1999).
[CrossRef] [PubMed]

J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[CrossRef] [PubMed]

A. A. Gerencser, J. Doczi, B. Töröcsik, E. Bossy-Wetzel, and V. Adam-Vizi, “Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte-neuron differences,” Biophys. J. 95(5), 2583–2598 (2008).
[CrossRef] [PubMed]

P. H. Wu, S. H. Arce, P. R. Burney, and Y. Tseng, “A novel approach to high accuracy of video-based microrheology,” Biophys. J. 96(12), 5103–5111 (2009).
[CrossRef] [PubMed]

T. Savin and P. S. Doyle, “Static and dynamic errors in particle tracking microrheology,” Biophys. J. 88(1), 623–638 (2005).
[CrossRef]

Eur. Phys. J. D

Y. Reibel, M. Jung, M. Bouhifd, B. Cunin, and C. Draman, “CCD or CMOS Camera Noise Characterisation,” Eur. Phys. J. D 21, 75–80 (2003).

IEEE Trans. Electron. Dev.

J. Hynecek, “Impactron-a new solid state image intensifier,” IEEE Trans. Electron. Dev. 48(10), 2238–2241 (2001).
[CrossRef]

M. S. Robbins and B. J. Hadwen, “The noise performance of electron multiplying charge-coupled devices,” IEEE Trans. Electron. Dev. 50(5), 1227–1232 (2003).
[CrossRef]

J. Cell Sci.

S. Li, S. L. Lian, J. J. Moser, M. L. Fritzler, M. J. Fritzler, M. Satoh, and E. K. Chan, “Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing,” J. Cell Sci. 121(24), 4134–4144 (2008).
[CrossRef] [PubMed]

C. H. Eskiw, G. Dellaire, J. S. Mymryk, and D. P. Bazett-Jones, “Size, position and dynamic behavior of PML nuclear bodies following cell stress as a paradigm for supramolecular trafficking and assembly,” J. Cell Sci. 116(21), 4455–4466 (2003).
[CrossRef] [PubMed]

S. Weidtkamp-Peters, T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich, and P. Hemmerich, “Dynamics of component exchange at PML nuclear bodies,” J. Cell Sci. 121(16), 2731–2743 (2008).
[CrossRef] [PubMed]

J. Phys. B

L. Zhang, L. Neves, J. S. Lundeen, and I. A. Walmsley, “A Characterization of the Single-photon Sensitivity of an Electron Multiplying Charge-Coupled Device,” J. Phys. B 42(11), 114011 (2009).
[CrossRef]

Mol. Biol. Cell

T. P. Kole, Y. Tseng, L. Huang, J. L. Katz, and D. Wirtz, “Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation,” Mol. Biol. Cell 15(7), 3475–3484 (2004).
[CrossRef] [PubMed]

Nat. Cell Biol.

G. Fink, L. Hajdo, K. J. Skowronek, C. Reuther, A. A. Kasprzak, and S. Diez, “The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding,” Nat. Cell Biol. 11(6), 717–723 (2009).
[CrossRef] [PubMed]

M. Benoit, D. Gabriel, G. Gerisch, and H. E. Gaub, “Discrete interactions in cell adhesion measured by single-molecule force spectroscopy,” Nat. Cell Biol. 2(6), 313–317 (2000).
[CrossRef] [PubMed]

Nat. Protoc.

E. S. Levitan, F. Lanni, and D. Shakiryanova, “In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction,” Nat. Protoc. 2(5), 1117–1125 (2007).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A.

S. M. Görisch, M. Wachsmuth, C. Ittrich, C. P. Bacher, K. Rippe, and P. Lichter, “Nuclear body movement is determined by chromatin accessibility and dynamics,” Proc. Natl. Acad. Sci. U.S.A. 101(36), 13221–13226 (2004).
[CrossRef] [PubMed]

Science

C. Bakal, J. Aach, G. Church, and N. Perrimon, “Quantitative morphological signatures define local signaling networks regulating cell morphology,” Science 316(5832), 1753–1756 (2007).
[CrossRef] [PubMed]

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, “Kinesin walks hand-over-hand,” Science 303(5658), 676–678 (2004).
[CrossRef]

SPIE Image Acquis. Sci. Imaging Syst.

J. C. Mullikin, L. J. van Vliet, H. Netten, F. R. Boddeke, G. van der Feltz, and I. T. Young, “Methods For CCD Camera Characterization,” SPIE Image Acquis. Sci. Imaging Syst. 2173, 73–84 (1994).

Other

R. C. Gonzalez, and R. E. Woods, Digital Image Processing (Prentice Hall, Upper Saddle River, NJ, 2002).

I. McWhirter, “Electron Multiplying CCDs - New Technology for Low Light Level Imaging,” Proceedings of 33rd annual European meeting on atmospheric studies by optical methods IRF science report 292, 61–66 (2008).

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Figures (6)

Fig. 1
Fig. 1

EM gain causes contradictory effects on the spatial resolution of protein clusters. An NIH 3T3 fibroblast expressed two different proteins, GFP-Ago2 and YFP-Sumo1, forming two types of protein complexes containing one kind of fusion protein each. These speckle-like complexes (n = 6) were tracked to analyze the positioning error after the cell was fixed by formaldehyde. The analyzed results are listed in the table beside the image with the object numbers designated in the image. The effects of EM gain (at k = 2000) on the resolution (presented by the variance of tracked positions) was listed as the ratio of the positioning resolution at k = 0 to k = 2000 (far right column).

Fig. 2
Fig. 2

EM gain parameters are determined experimentally. A. Schematic plot of photon conversion and noise propagation in the EMCCD camera. B. Logarithmic plot of intensity count ( I o u t , k ) at different EM gain (▲) versus I o u t , 0 under varying light intensities. The solid line represents the least squared fitting results to the linear model. Actual electron multiplication gain can be estimated from the slope of fitted model. C. Scatter plot of σ o u t , k 2 versus I o u t , k at k = 0, 1000, 2000, 3000 and 4000 in the logarithmic scale. Linear model fitting results at different EM gain are shown as a solid line and agree well with experimental data. R2 > 0.99 for all fitted models.

Fig. 3
Fig. 3

The optimal setting of the EM gain depends on the intensity of the signal. A. The correlation between SNR and I o u t , k is plotted for k = 2000. The theoretically derived SNR curve (solid line) agrees well with experimental data (▲). B. Values of the SNR at different EM gain (k = 0, 1000, 2000, 3000 and 4000) are determined theoretically and plotted at the same amount of light intensity as I o u t , 0 . At a high incident light intensity, the highest SNR is at k = 0, and the SNR decreases with increasing k. Inset: the region marked by the dashed box in panel B is shown in more detail. The arrow points in the direction of increasing k. C. The SNR crossover between k = 0 and a given k occurs around I o u t , 0 = 1800 au.

Fig. 4
Fig. 4

The relation of the k and ε S is affected by I p e a k . A. The EM gain setting affects the ε S dependency on I p e a k . These results show that EM gain effectively improves the ε S at low signal intensity, but the effect diminishes with increasing I p e a k of the objects. After the ε S at a certain k crosses over that of k = 0 ( I p e a k ~2000 au), a contrary effect occurs. B. The crossover points of the peak intensity at the junction of the positioning error curves of non-zero k and at k = 0 (●) depend on k. The crossover points set an upper bound as the critical points for the highest I p e a k values at a certain EM gain; at I p e a k < I C , k , one can still take the advantage of EM gain to reduce the positioning error in particle tracking experiments. The error bar is the standard deviation from five individual simulation results.

Fig. 5
Fig. 5

The EM gain performance in particle tracking depends on experimental conditions. A. Background intensity, I B G , affects the I C , k significantly. With I B G > ~900 au, the EM gain will not improve the ε S for particle tracking. B. A large apparent radius of particle, Ra , also affects the I C , k significantly. Open triangles (△) and open squares (□) correspond to k = 1000 and 2000, respectively.

Fig. 6
Fig. 6

Plot of ε S , 1000 vs. ε S , 0 . Two experimental conditions were chosen: 35 microspheres at high I p e a k at 100% light source intensity (□) and 43 microspheres at low I p e a k at 25% power of light source intensity (●). The solid line corresponds to ε S , 1000 = ε S , 0 . Zero EM gain causes a 23% ( ± 10%) improvement in positioning error at 100% light source intensity in which I p e a k is larger than 10,000 au and background intensity is ~5000 au. On the other hand, EM gain (k = 1000) improved the positioning error by 46% ( ± 11%) for the particle with small I p e a k (~1000 au) and minimal background ( I B G ~0 at 25% light source intensity).

Tables (2)

Tables Icon

Table 1 Summary of Notation

Tables Icon

Table 2 The values of the effective EM gain (k), excess factor (F), intensity offset ( I o f f s e t ), and relative working range at several EM gain settings estimated for our system.

Equations (7)

Equations on this page are rendered with MathJax. Learn more.

log I P ( x , y ) = log ( I ) ( x μ x ) 2 + ( y μ y ) 2 2 × R a 2 ,
I o u t , k = ( N P E + N D E ) × G E M , k × G A D C + I o f f s e t , k .
( N P E + N D E ) = ( I o u t , 0 I o f f s e t , 0 ) / ( G E M , 0 × G A D C ) .
I o u t , k = G E M , k × I o u t , 0 + I o f f s e t , k G E M , k × I o f f s e t , 0 .
σ o u t , k 2 = ( N P E + N D E ) × G E M , k 2 × G A D C 2 × F k 2 + σ R 2 .
σ o u t , k 2 = I o u t , k × G E M , k × G A D C × F k 2 + σ R 2 I o f f s e t , k × G E M , k × G A D C × F k 2 .
S N R = N P E × G E M , k × G A D C / ( ( N P E + N D E ) × G E M , k 2 × G A D C 2 × F k 2 + σ R 2 ) .

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