Abstract

The ability to detect single molecules over the electronic noise requires high performance detector systems. Electron Multiplying Charge-Coupled Device (EMCCD) cameras have been employed successfully to image single molecules. Recently, scientific Complementary Metal Oxide Semiconductor (sCMOS) based cameras have been introduced with very low read noise at faster read out rates, smaller pixel sizes and a lower price compared to EMCCD cameras. In this study, we have compared the two technologies using two EMCCD and three sCMOS cameras to detect single Cy5 molecules. Our findings indicate that the sCMOS cameras perform similar to EMCCD cameras for detecting and localizing single Cy5 molecules.

© 2012 OSA

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2011

2010

A. Edelstein,, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μManager,” Curr. Protoc. Mol. Biol. 14, 1–17 (2010).

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, “Fast, single-molecule localization that achieves theoretically minimum uncertainty,” Nat. Methods 7(5), 373–375 (2010).
[CrossRef] [PubMed]

2006

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J. 37(5), 433–451 (2006).
[CrossRef]

E. G. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

2005

2004

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J. 86(2), 1185–1200 (2004).
[CrossRef] [PubMed]

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
[CrossRef] [PubMed]

2003

W. E. Moerner and D. P. Fromm, “Methods of single-molecule fluorescence spectroscopy and microscopy,” Rev. Sci. Instrum. 74(8), 3597–3619 (2003).
[CrossRef]

X. Michalet, A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft, and S. Weiss, “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct. 32(1), 161–182 (2003).
[CrossRef] [PubMed]

2002

D. Joseph and S. Collins, “Modeling, calibration, and correction of nonlinear illumination-dependent fixed pattern noise in logarithmic CMOS image sensors,” IEEE Trans. Instrum. Meas. 51(5), 996–1001 (2002).
[CrossRef]

2001

M. K. Cheezum, W. F. Walker, and W. H. Guilford, “Quantitative comparison of algorithms for tracking single fluorescent particles,” Biophys. J. 81(4), 2378–2388 (2001).
[CrossRef] [PubMed]

2000

A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
[CrossRef] [PubMed]

1997

G. J. Schütz, H. Schindler, and T. Schmidt, “Single-molecule microscopy on model membranes reveals anomalous diffusion,” Biophys. J. 73(2), 1073–1080 (1997).
[CrossRef] [PubMed]

1994

J. K. Trautman, J. J. Macklin, L. E. Brus, and E. Betzig, “Near-field spectroscopy of single molecules at room temperature,” Nature 369(6475), 40–42 (1994).
[CrossRef]

1989

L. A. Ernst, R. K. Gupta, R. B. Mujumdar, and A. S. Waggoner, “Cyanine dye labeling reagents for sulfhydryl groups,” Cytometry 10(1), 3–10 (1989).
[CrossRef] [PubMed]

Aguet, F.

Amodaj, N.

A. Edelstein,, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μManager,” Curr. Protoc. Mol. Biol. 14, 1–17 (2010).

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Beligere, G. S.

A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
[CrossRef] [PubMed]

Betzig, E.

J. K. Trautman, J. J. Macklin, L. E. Brus, and E. Betzig, “Near-field spectroscopy of single molecules at room temperature,” Nature 369(6475), 40–42 (1994).
[CrossRef]

Betzig, E. G.

E. G. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bigas, M.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J. 37(5), 433–451 (2006).
[CrossRef]

Bonifacino, J. S.

E. G. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Brus, L. E.

J. K. Trautman, J. J. Macklin, L. E. Brus, and E. Betzig, “Near-field spectroscopy of single molecules at room temperature,” Nature 369(6475), 40–42 (1994).
[CrossRef]

Cabruja, E.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J. 37(5), 433–451 (2006).
[CrossRef]

Cheezum, M. K.

M. K. Cheezum, W. F. Walker, and W. H. Guilford, “Quantitative comparison of algorithms for tracking single fluorescent particles,” Biophys. J. 81(4), 2378–2388 (2001).
[CrossRef] [PubMed]

Chemla, D. S.

A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
[CrossRef] [PubMed]

Collins, S.

D. Joseph and S. Collins, “Modeling, calibration, and correction of nonlinear illumination-dependent fixed pattern noise in logarithmic CMOS image sensors,” IEEE Trans. Instrum. Meas. 51(5), 996–1001 (2002).
[CrossRef]

Dahan, M.

A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
[CrossRef] [PubMed]

Davidson, M. W.

E. G. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Dawson, P. E.

A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
[CrossRef] [PubMed]

Deniz, A. A.

A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
[CrossRef] [PubMed]

Ding, J.

Doose, S.

X. Michalet, A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft, and S. Weiss, “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct. 32(1), 161–182 (2003).
[CrossRef] [PubMed]

Edelstein,, A.

A. Edelstein,, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μManager,” Curr. Protoc. Mol. Biol. 14, 1–17 (2010).

Ernst, L. A.

L. A. Ernst, R. K. Gupta, R. B. Mujumdar, and A. S. Waggoner, “Cyanine dye labeling reagents for sulfhydryl groups,” Cytometry 10(1), 3–10 (1989).
[CrossRef] [PubMed]

Forest, J.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J. 37(5), 433–451 (2006).
[CrossRef]

Fromm, D. P.

W. E. Moerner and D. P. Fromm, “Methods of single-molecule fluorescence spectroscopy and microscopy,” Rev. Sci. Instrum. 74(8), 3597–3619 (2003).
[CrossRef]

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(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Guilford, W. H.

M. K. Cheezum, W. F. Walker, and W. H. Guilford, “Quantitative comparison of algorithms for tracking single fluorescent particles,” Biophys. J. 81(4), 2378–2388 (2001).
[CrossRef] [PubMed]

Gupta, R. K.

L. A. Ernst, R. K. Gupta, R. B. Mujumdar, and A. S. Waggoner, “Cyanine dye labeling reagents for sulfhydryl groups,” Cytometry 10(1), 3–10 (1989).
[CrossRef] [PubMed]

Hess, H. F.

E. G. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Hoover, K.

A. Edelstein,, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μManager,” Curr. Protoc. Mol. Biol. 14, 1–17 (2010).

Huang, Z.-L.

Ivanchenko, S.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
[CrossRef] [PubMed]

Joseph, D.

D. Joseph and S. Collins, “Modeling, calibration, and correction of nonlinear illumination-dependent fixed pattern noise in logarithmic CMOS image sensors,” IEEE Trans. Instrum. Meas. 51(5), 996–1001 (2002).
[CrossRef]

Joseph, N.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, “Fast, single-molecule localization that achieves theoretically minimum uncertainty,” Nat. Methods 7(5), 373–375 (2010).
[CrossRef] [PubMed]

Kapanidis, A. N.

X. Michalet, A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft, and S. Weiss, “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct. 32(1), 161–182 (2003).
[CrossRef] [PubMed]

Laurence, T.

X. Michalet, A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft, and S. Weiss, “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct. 32(1), 161–182 (2003).
[CrossRef] [PubMed]

Laurence, T. A.

A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
[CrossRef] [PubMed]

Lidke, K. A.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, “Fast, single-molecule localization that achieves theoretically minimum uncertainty,” Nat. Methods 7(5), 373–375 (2010).
[CrossRef] [PubMed]

Lindwasser, O. W.

E. G. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

E. G. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Liu, Y.

Long, F.

Luo, Q.

Ma, H.

Macklin, J. J.

J. K. Trautman, J. J. Macklin, L. E. Brus, and E. Betzig, “Near-field spectroscopy of single molecules at room temperature,” Nature 369(6475), 40–42 (1994).
[CrossRef]

Martin, A. B.

A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
[CrossRef] [PubMed]

Mason, M. D.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Michalet, X.

X. Michalet, A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft, and S. Weiss, “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct. 32(1), 161–182 (2003).
[CrossRef] [PubMed]

Moerner, W. E.

W. E. Moerner and D. P. Fromm, “Methods of single-molecule fluorescence spectroscopy and microscopy,” Rev. Sci. Instrum. 74(8), 3597–3619 (2003).
[CrossRef]

Mujumdar, R. B.

L. A. Ernst, R. K. Gupta, R. B. Mujumdar, and A. S. Waggoner, “Cyanine dye labeling reagents for sulfhydryl groups,” Cytometry 10(1), 3–10 (1989).
[CrossRef] [PubMed]

Nienhaus, G. U.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
[CrossRef] [PubMed]

Ober, R. J.

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J. 86(2), 1185–1200 (2004).
[CrossRef] [PubMed]

Olenych, S.

E. G. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Oswald, F.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
[CrossRef] [PubMed]

Patterson, G. H.

E. G. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Pflughoefft, M.

X. Michalet, A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft, and S. Weiss, “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct. 32(1), 161–182 (2003).
[CrossRef] [PubMed]

Pinaud, F.

X. Michalet, A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft, and S. Weiss, “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct. 32(1), 161–182 (2003).
[CrossRef] [PubMed]

Qin, L.

Ram, S.

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J. 86(2), 1185–1200 (2004).
[CrossRef] [PubMed]

Rieger, B.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, “Fast, single-molecule localization that achieves theoretically minimum uncertainty,” Nat. Methods 7(5), 373–375 (2010).
[CrossRef] [PubMed]

Roecker, C.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
[CrossRef] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Salih, A.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
[CrossRef] [PubMed]

Salvi, J.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J. 37(5), 433–451 (2006).
[CrossRef]

Schindler, H.

G. J. Schütz, H. Schindler, and T. Schmidt, “Single-molecule microscopy on model membranes reveals anomalous diffusion,” Biophys. J. 73(2), 1073–1080 (1997).
[CrossRef] [PubMed]

Schmidt, T.

G. J. Schütz, H. Schindler, and T. Schmidt, “Single-molecule microscopy on model membranes reveals anomalous diffusion,” Biophys. J. 73(2), 1073–1080 (1997).
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Schmitt, F.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
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A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
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G. J. Schütz, H. Schindler, and T. Schmidt, “Single-molecule microscopy on model membranes reveals anomalous diffusion,” Biophys. J. 73(2), 1073–1080 (1997).
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Smith, C. S.

C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, “Fast, single-molecule localization that achieves theoretically minimum uncertainty,” Nat. Methods 7(5), 373–375 (2010).
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E. G. 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(5793), 1642–1645 (2006).
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J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
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A. Edelstein,, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μManager,” Curr. Protoc. Mol. Biol. 14, 1–17 (2010).

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J. K. Trautman, J. J. Macklin, L. E. Brus, and E. Betzig, “Near-field spectroscopy of single molecules at room temperature,” Nature 369(6475), 40–42 (1994).
[CrossRef]

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Vale, R.

A. Edelstein,, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μManager,” Curr. Protoc. Mol. Biol. 14, 1–17 (2010).

Van De Ville, D.

Waggoner, A. S.

L. A. Ernst, R. K. Gupta, R. B. Mujumdar, and A. S. Waggoner, “Cyanine dye labeling reagents for sulfhydryl groups,” Cytometry 10(1), 3–10 (1989).
[CrossRef] [PubMed]

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M. K. Cheezum, W. F. Walker, and W. H. Guilford, “Quantitative comparison of algorithms for tracking single fluorescent particles,” Biophys. J. 81(4), 2378–2388 (2001).
[CrossRef] [PubMed]

Ward, E. S.

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J. 86(2), 1185–1200 (2004).
[CrossRef] [PubMed]

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X. Michalet, A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft, and S. Weiss, “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct. 32(1), 161–182 (2003).
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A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
[CrossRef] [PubMed]

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J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
[CrossRef] [PubMed]

Zeng, S.

Zhang, Z.

Zhu, H.

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Annu. Rev. Biophys. Biomol. Struct.

X. Michalet, A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft, and S. Weiss, “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct. 32(1), 161–182 (2003).
[CrossRef] [PubMed]

Biophys. J.

G. J. Schütz, H. Schindler, and T. Schmidt, “Single-molecule microscopy on model membranes reveals anomalous diffusion,” Biophys. J. 73(2), 1073–1080 (1997).
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S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
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M. K. Cheezum, W. F. Walker, and W. H. Guilford, “Quantitative comparison of algorithms for tracking single fluorescent particles,” Biophys. J. 81(4), 2378–2388 (2001).
[CrossRef] [PubMed]

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J. 86(2), 1185–1200 (2004).
[CrossRef] [PubMed]

Curr. Protoc. Mol. Biol.

A. Edelstein,, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μManager,” Curr. Protoc. Mol. Biol. 14, 1–17 (2010).

Cytometry

L. A. Ernst, R. K. Gupta, R. B. Mujumdar, and A. S. Waggoner, “Cyanine dye labeling reagents for sulfhydryl groups,” Cytometry 10(1), 3–10 (1989).
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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(10), 793–796 (2006).
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C. S. Smith, N. Joseph, B. Rieger, and K. A. Lidke, “Fast, single-molecule localization that achieves theoretically minimum uncertainty,” Nat. Methods 7(5), 373–375 (2010).
[CrossRef] [PubMed]

Nature

J. K. Trautman, J. J. Macklin, L. E. Brus, and E. Betzig, “Near-field spectroscopy of single molecules at room temperature,” Nature 369(6475), 40–42 (1994).
[CrossRef]

Opt. Express

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

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Roecker, A. Salih, K. Spindler, and G. U. Nienhaus, “EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion,” Proc. Natl. Acad. Sci. U.S.A. 101(45), 15905–15910 (2004).
[CrossRef] [PubMed]

A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A. 97(10), 5179–5184 (2000).
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D. Toomre and J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006), Chap. 10.

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Figures (8)

Fig. 7
Fig. 7

(A) Single Cy5 molecules imaged using the various sensors used in this study (scale bar = 200nm). (B) Single Cy5 molecules sample density imaged using Evolve 512 EMCCD camera (scale bar = 9µm).

Fig. 1
Fig. 1

Distribution of peak intensity for single Cy5 molecules detected using, (A) Evolve 512 EMCCD, (B) Andor 887 EMCCD, (C) pco.Edge sCMOS, (D) Andor Neo sCMOS, and (E) Hamamatsu ORCA Flash 2.8.

Fig. 2
Fig. 2

Distribution of peak signal-to-noise ratio for single Cy5 molecules imaged using (A) Evolve 512 EMCCD, (B) Andor 887 EMCCD, (C) pco.Edge sCMOS, (D) Andor Neo sCMOS, and (E) Hamamatsu ORCA Flash 2.8.

Fig. 3
Fig. 3

Distribution of the standard error in position (Δr) values for non-linear least squares fitting of the PSF for single Cy5 molecules imaged using (A) Evolve 512 EMCCD, (B) Andor 887 EMCCD, (C) pco.Edge sCMOS, (D) Andor Neo sCMOS, and (E) Hamamatsu ORCA Flash 2.8.

Fig. 4
Fig. 4

Distribution of standard deviation in localization precision values for MLE fitting (σMLE) of the PSF for single Cy5 molecules imaged using (A) Evolve 512 EMCCD, (B) Andor 887 EMCCD, (C) pco.Edge sCMOS, (D) Andor Neo sCMOS, and (E) Hamamatsu ORCA Flash 2.8.

Fig. 5
Fig. 5

A heat map showing the dependence of Δr values for non-linear least squares fitting on integrated object intensity for single Cy5 molecules imaged using (A) Evolve 512 EMCCD, (B) Andor 887 EMCCD, (C) pco.Edge sCMOS, (D) Andor Neo sCMOS, and (E) Hamamatsu ORCA Flash 2.8. The circles are the simulated values for three different integrated photon counts per object (100, 200, 500).

Fig. 8
Fig. 8

Simulated single molecule images for (A) EMCCD sensor, (B) an sCMOS sensor used in pco.Edge and Andor Neo cameras, and (C) an sCMOS sensor used in the Hamamatsu ORCA Flash 2.8 camera (N = 100 photons). The lower panel shows experimental data for the same sensors respectively: (D) EMCCD image taken using Andor 887, (E) sCMOS image taken using pco.Edge, (F) Image taken using Hamamatsu ORCA Flash2.8. The scale bars are each 200 nm.

Fig. 6
Fig. 6

Simulation results showing expected standard error in position (Δr) for a sCMOS detector as a function of pixel size (Mean and median values) from nonlinear least squares fitting to a Gaussian PSF. The simulations were performed for 200 photons.

Tables (1)

Tables Icon

Table 1 Key characteristics of the cameras used in the study

Equations (7)

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S N R = ( I 0 I B A r e a ) / σ B
I ( x , y ) = I B + I 0 e x p [ ( x μ x ) 2 2 σ x 2 ( y μ y ) 2 2 σ y 2 ]
v a r ( θ ^ ) I ( θ ) 1
I ( θ ) i j = E [ l n ( L ( x | θ ) ) θ i . l n ( L ( x | θ ) ) θ j ]
I M S E = E [ f ^ ( x ) f ( x ) ] 2 d x
h n = ( 6 1 / 3 ( f ' ( x ) 2 d x ) 1 / 3 ) / n 3
N b i n = ( max ( D ) min ( D ) ) / h n

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