Abstract

In the community of localization-based super-resolution microscopy (or called localization microscopy), it is generally believed that the emission of single molecules is so weak that an EMCCD (electron multiplying charge coupled device) camera is necessary to be used as the detector by eliminating read noise. Here we evaluate the possibility of a new kind of low light detector, scientific complementary metal-oxide-semiconductor (sCMOS) camera in localization microscopy. We demonstrate experimentally that sCMOS is capable of imaging actin bundles with FWHM diameter of 37 nm, evidencing the capability of sCMOS in localization microscopy. We further characterize the noise performance of sCMOS and find out that, with the use of a bright fluorescence probe such as d2EosFP, localization microscopy imaging is now working in the shot noise limited region.

© 2011 OSA

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2011 (2)

2010 (3)

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[CrossRef]

T. W. Quan, S. Q. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt. 15(6), 066005 (2010).
[CrossRef] [PubMed]

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(5), 377–381 (2010).
[CrossRef] [PubMed]

2009 (3)

P. N. Hedde, J. Fuchs, F. Oswald, J. Wiedenmann, and G. U. Nienhaus, “Online image analysis software for photoactivation localization microscopy,” Nat. Methods 6(10), 689–690 (2009).
[CrossRef] [PubMed]

J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol. 19(11), 555–565 (2009).
[CrossRef] [PubMed]

B. Huang, M. Bates, and X. W. Zhuang, “Super-resolution fluorescence microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

2008 (1)

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol. 9(12), 929–943 (2008).
[CrossRef] [PubMed]

2007 (1)

X. Michalet, O. H. W. Siegmund, J. V. Vallerga, P. Jelinsky, J. E. Millaud, and S. Weiss, “Detectors for single-molecule fluorescence imaging and spectroscopy,” J. Mod. Opt. 54(2-3), 239–281 (2007).
[CrossRef] [PubMed]

2006 (5)

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(5793), 1642–1645 (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]

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

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

J. Wiedenmann and G. U. Nienhaus, “Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family,” Expert Rev. Proteomics 3(3), 361–374 (2006).
[CrossRef] [PubMed]

2005 (1)

K. Nienhaus, G. U. Nienhaus, J. Wiedenmann, and H. Nar, “Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP,” Proc. Natl. Acad. Sci. U.S.A. 102(26), 9156–9159 (2005).
[CrossRef] [PubMed]

2003 (2)

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

Appelbaum, J.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[CrossRef]

Balicki, J.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[CrossRef]

Bates, M.

B. Huang, M. Bates, and X. W. Zhuang, “Super-resolution fluorescence microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

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

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(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. 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]

Cabruja, E.

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

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(5), 377–381 (2010).
[CrossRef] [PubMed]

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

Ding, J. P.

Do, H.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[CrossRef]

Fernández-Suárez, M.

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol. 9(12), 929–943 (2008).
[CrossRef] [PubMed]

Flyvbjerg, H.

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(5), 377–381 (2010).
[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]

Fowler, B.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[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]

Fuchs, J.

P. N. Hedde, J. Fuchs, F. Oswald, J. Wiedenmann, and G. U. Nienhaus, “Online image analysis software for photoactivation localization microscopy,” Nat. Methods 6(10), 689–690 (2009).
[CrossRef] [PubMed]

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]

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]

He, J.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods 8(6), 499–505 (2011).
[CrossRef] [PubMed]

Hedde, P. N.

P. N. Hedde, J. Fuchs, F. Oswald, J. Wiedenmann, and G. U. Nienhaus, “Online image analysis software for photoactivation localization microscopy,” Nat. Methods 6(10), 689–690 (2009).
[CrossRef] [PubMed]

Hess, H. F.

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(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]

Huang, B.

B. Huang, M. Bates, and X. W. Zhuang, “Super-resolution fluorescence microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

Huang, Z. L.

T. W. Quan, H. Y. Zhu, X. M. Liu, Y. F. Liu, J. P. Ding, S. Q. Zeng, and Z. L. Huang, “High-density localization of active molecules using Structured Sparse Model and Bayesian Information Criterion,” Opt. Express 19(18), 16963–16974 (2011).
[CrossRef] [PubMed]

T. W. Quan, S. Q. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt. 15(6), 066005 (2010).
[CrossRef] [PubMed]

Jelinsky, P.

X. Michalet, O. H. W. Siegmund, J. V. Vallerga, P. Jelinsky, J. E. Millaud, and S. Weiss, “Detectors for single-molecule fluorescence imaging and spectroscopy,” J. Mod. Opt. 54(2-3), 239–281 (2007).
[CrossRef] [PubMed]

Jones, S. A.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods 8(6), 499–505 (2011).
[CrossRef] [PubMed]

Li, W.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[CrossRef]

Lindwasser, O. 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol. 19(11), 555–565 (2009).
[CrossRef] [PubMed]

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

Liu, C. A.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[CrossRef]

Liu, X. M.

Liu, Y. F.

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, O. H. W. Siegmund, J. V. Vallerga, P. Jelinsky, J. E. Millaud, and S. Weiss, “Detectors for single-molecule fluorescence imaging and spectroscopy,” J. Mod. Opt. 54(2-3), 239–281 (2007).
[CrossRef] [PubMed]

Millaud, J. E.

X. Michalet, O. H. W. Siegmund, J. V. Vallerga, P. Jelinsky, J. E. Millaud, and S. Weiss, “Detectors for single-molecule fluorescence imaging and spectroscopy,” J. Mod. Opt. 54(2-3), 239–281 (2007).
[CrossRef] [PubMed]

Mims, S.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[CrossRef]

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]

Mortensen, K. I.

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(5), 377–381 (2010).
[CrossRef] [PubMed]

Nar, H.

K. Nienhaus, G. U. Nienhaus, J. Wiedenmann, and H. Nar, “Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP,” Proc. Natl. Acad. Sci. U.S.A. 102(26), 9156–9159 (2005).
[CrossRef] [PubMed]

Nienhaus, G. U.

P. N. Hedde, J. Fuchs, F. Oswald, J. Wiedenmann, and G. U. Nienhaus, “Online image analysis software for photoactivation localization microscopy,” Nat. Methods 6(10), 689–690 (2009).
[CrossRef] [PubMed]

J. Wiedenmann and G. U. Nienhaus, “Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family,” Expert Rev. Proteomics 3(3), 361–374 (2006).
[CrossRef] [PubMed]

K. Nienhaus, G. U. Nienhaus, J. Wiedenmann, and H. Nar, “Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP,” Proc. Natl. Acad. Sci. U.S.A. 102(26), 9156–9159 (2005).
[CrossRef] [PubMed]

Nienhaus, K.

K. Nienhaus, G. U. Nienhaus, J. Wiedenmann, and H. Nar, “Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP,” Proc. Natl. Acad. Sci. U.S.A. 102(26), 9156–9159 (2005).
[CrossRef] [PubMed]

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

Oswald, F.

P. N. Hedde, J. Fuchs, F. Oswald, J. Wiedenmann, and G. U. Nienhaus, “Online image analysis software for photoactivation localization microscopy,” Nat. Methods 6(10), 689–690 (2009).
[CrossRef] [PubMed]

Patterson, G. H.

J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol. 19(11), 555–565 (2009).
[CrossRef] [PubMed]

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

Quan, T. W.

T. W. Quan, H. Y. Zhu, X. M. Liu, Y. F. Liu, J. P. Ding, S. Q. Zeng, and Z. L. Huang, “High-density localization of active molecules using Structured Sparse Model and Bayesian Information Criterion,” Opt. Express 19(18), 16963–16974 (2011).
[CrossRef] [PubMed]

T. W. Quan, S. Q. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt. 15(6), 066005 (2010).
[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]

Rust, M. J.

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

Shim, S. H.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods 8(6), 499–505 (2011).
[CrossRef] [PubMed]

Siegmund, O. H. W.

X. Michalet, O. H. W. Siegmund, J. V. Vallerga, P. Jelinsky, J. E. Millaud, and S. Weiss, “Detectors for single-molecule fluorescence imaging and spectroscopy,” J. Mod. Opt. 54(2-3), 239–281 (2007).
[CrossRef] [PubMed]

Sougrat, R.

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

Spudich, J. A.

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(5), 377–381 (2010).
[CrossRef] [PubMed]

Ting, A. Y.

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol. 9(12), 929–943 (2008).
[CrossRef] [PubMed]

Vallerga, J. V.

X. Michalet, O. H. W. Siegmund, J. V. Vallerga, P. Jelinsky, J. E. Millaud, and S. Weiss, “Detectors for single-molecule fluorescence imaging and spectroscopy,” J. Mod. Opt. 54(2-3), 239–281 (2007).
[CrossRef] [PubMed]

Vu, P.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[CrossRef]

Weiss, S.

X. Michalet, O. H. W. Siegmund, J. V. Vallerga, P. Jelinsky, J. E. Millaud, and S. Weiss, “Detectors for single-molecule fluorescence imaging and spectroscopy,” J. Mod. Opt. 54(2-3), 239–281 (2007).
[CrossRef] [PubMed]

Wiedenmann, J.

P. N. Hedde, J. Fuchs, F. Oswald, J. Wiedenmann, and G. U. Nienhaus, “Online image analysis software for photoactivation localization microscopy,” Nat. Methods 6(10), 689–690 (2009).
[CrossRef] [PubMed]

J. Wiedenmann and G. U. Nienhaus, “Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family,” Expert Rev. Proteomics 3(3), 361–374 (2006).
[CrossRef] [PubMed]

K. Nienhaus, G. U. Nienhaus, J. Wiedenmann, and H. Nar, “Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP,” Proc. Natl. Acad. Sci. U.S.A. 102(26), 9156–9159 (2005).
[CrossRef] [PubMed]

Zeng, S. Q.

T. W. Quan, H. Y. Zhu, X. M. Liu, Y. F. Liu, J. P. Ding, S. Q. Zeng, and Z. L. Huang, “High-density localization of active molecules using Structured Sparse Model and Bayesian Information Criterion,” Opt. Express 19(18), 16963–16974 (2011).
[CrossRef] [PubMed]

T. W. Quan, S. Q. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt. 15(6), 066005 (2010).
[CrossRef] [PubMed]

Zhu, H. Y.

Zhuang, X.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods 8(6), 499–505 (2011).
[CrossRef] [PubMed]

Zhuang, X. W.

B. Huang, M. Bates, and X. W. Zhuang, “Super-resolution fluorescence microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

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

Annu. Rev. Biochem. (1)

B. Huang, M. Bates, and X. W. Zhuang, “Super-resolution fluorescence microscopy,” Annu. Rev. Biochem. 78(1), 993–1016 (2009).
[CrossRef] [PubMed]

Biophys. J. (1)

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]

Expert Rev. Proteomics (1)

J. Wiedenmann and G. U. Nienhaus, “Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family,” Expert Rev. Proteomics 3(3), 361–374 (2006).
[CrossRef] [PubMed]

IEEE Trans. Electron. Dev. (1)

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. Biomed. Opt. (1)

T. W. Quan, S. Q. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt. 15(6), 066005 (2010).
[CrossRef] [PubMed]

J. Mod. Opt. (1)

X. Michalet, O. H. W. Siegmund, J. V. Vallerga, P. Jelinsky, J. E. Millaud, and S. Weiss, “Detectors for single-molecule fluorescence imaging and spectroscopy,” J. Mod. Opt. 54(2-3), 239–281 (2007).
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Microelectron. J. (1)

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J. 37(5), 433–451 (2006).
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Nat. Methods (4)

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

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(5), 377–381 (2010).
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P. N. Hedde, J. Fuchs, F. Oswald, J. Wiedenmann, and G. U. Nienhaus, “Online image analysis software for photoactivation localization microscopy,” Nat. Methods 6(10), 689–690 (2009).
[CrossRef] [PubMed]

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods 8(6), 499–505 (2011).
[CrossRef] [PubMed]

Nat. Rev. Mol. Cell Biol. (1)

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol. 9(12), 929–943 (2008).
[CrossRef] [PubMed]

Opt. Express (1)

Proc. Natl. Acad. Sci. U.S.A. (1)

K. Nienhaus, G. U. Nienhaus, J. Wiedenmann, and H. Nar, “Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP,” Proc. Natl. Acad. Sci. U.S.A. 102(26), 9156–9159 (2005).
[CrossRef] [PubMed]

Proc. SPIE (1)

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE 7536, 753607, 753607-12 (2010).
[CrossRef]

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W. E. Moerner and D. P. Fromm, “Methods of single-molecule fluorescence spectroscopy and microscopy,” Rev. Sci. Instrum. 74(8), 3597–3619 (2003).
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Science (1)

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(5793), 1642–1645 (2006).
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Trends Cell Biol. (1)

J. Lippincott-Schwartz and G. H. Patterson, “Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging,” Trends Cell Biol. 19(11), 555–565 (2009).
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J. R. Janesick, Photon transfer: DN [lambda] (SPIE, Bellingham, Wash., 2007), pp. xiv, 258 p.

R. Yuste and A. Konnerth, Imaging in neuroscience and development: a laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2005).

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Figures (7)

Fig. 1
Fig. 1

Optical setup for localization microscopy imaging.

Fig. 2
Fig. 2

The optical setup for photon transfer curve (PTC) measurements

Fig. 3
Fig. 3

Comparison of the performance of the Flash 2.8 sCMOS camera (subset 1 in a, b, e, f and g) and the iXon 897 EMCCD camera (subset 2 in c, d, h, i and j) in TIRF microscopy (a, c) and localization microscopy (b, d) imaging of actin bundles in fixed HEK293T cells labeled with d2EosFP. A stack of 2000 image frames was firstly captured with the Flash2.8 sCMOS (Exposure time = 30 ms; Gain = 8; Binning = 2; In 184 x 104 pixels after binning; Effective pixel size at sample = 145 nm), and then another stack of 2000 image frames was obtained with the iXon 897 EMCCD (Exposure time = 30 ms; Gain = 120; No binning; In 167 x 94 pixels; Effective pixel size at sample = 160 nm). Note that the overall photon collection efficiency in these two camera ports is found to be almost identical. Histograms of total collected photons (e, h), signal-noise-ratio (f, i), and localization precision measured in standard deviation (g, j) from single molecules (Upper row: sCMOS; Lower row: EMCCD). The mean values of individual histograms are shown in the right corner of the corresponding figures. The total number of localized molecules is 68108 in (b) and 66353 in (d), respectively. Scale bars: 3 μm.

Fig. 4
Fig. 4

Representative PTCs for the Flash2.8 sCMOS. Note that: (a) For the PTC with Gain = 8 and Bin = 1 (top curve), the image frames (signal > 2200 photons) close to detector’s saturation are obtained by increasing exposure time from 50 ms to 100 ms because of the insufficient illumination intensity. (b) For the PTC with Gain = 1 and Bin = 2 (bottom curve), the image frames (signal > 11000 photons) close to detector’s saturation are obtained by increasing exposure time from 50 ms to 200 ms because of the insufficient illumination intensity. (c) These are representative curves selected from three repeated measurements, which gave almost identical results. (d) The PTC for Gain = 1 and Bin = 1 was not measured, because a much stronger illumination intensity necessary for this measurement could not be obtained from our tungsten-halogen light source. (e) The camera conversion factor, KADC(e/DN), is 0.609 e/DN for Gain = 8 & Bin = 1, 0.590 e/DN for Gain = 8 & Bin = 2, and 4.65 e/DN for Gain = 1 & Bin = 2, respectively. (f) Theoretical Shot Noise is calculated by the square root of Signal*QE [13], where the QE is the quantum efficiency (photon sensitivity) of the Flash2.8 sCMOS in 580 nm and is set to be 0.52.

Fig. 5
Fig. 5

Representative PTCs for the iXon 897 EMCCD. Note that (a) For the PTCs with EM Gain = 1 (top curve), the image frames (signal > 100000 photons) close to detector’s saturation are obtained by increasing exposure time from 50 ms to 120ms because of the insufficient illumination intensity. (b) These are representative curves selected from three repeated measurements, which gave almost identical results. (c) The camera conversion factor, KADC(e/DN), is 12.0 e/DN for M = 1, 0.107 e/DN for M = 120, and 0.0695 e/DN for M = 200, respectively. (d) The shot noise without noise factor (Shot Noise w/o Noise Factor) is from the Shot Noise w/ Noise Factor (that is, the shot noise in Section 2.4, Eq. (5) divided by the excess noise factor, which is 1 for M = 1, and 2 for M > 100, respectively [18]. (e) Theoretical Shot Noise was calculated by the square root of Signal*QE [13], where the QE is the quantum efficiency (photon sensitivity) of the iXon 897 EMCCD in 580 nm and is set to be 0.95.

Fig. 6
Fig. 6

The temporal read noise distribution of the Flash2.8 sCMOS. Note that the temporal read noise is reported to be 3.0 e- (rms) for Gain = 8 & Bin = 1 by the Vendor.

Fig. 7
Fig. 7

Comparison on the visibility of single molecule embedded in background noise by the Flash2.8 sCMOS (subset 1 in a, c and e) and the iXon 897 EMCCD (subset 2 in b, d and f). Histograms of the detected emission in the peak pixel after dark image offset subtraction (a, b), signal-background-ratio (c, d) from single molecules (Upper row: sCMOS; Lower row: EMCCD). Note that the histograms are obtained from analyzing Fig. 3b and Fig. 3d. The mean values of individual histograms are shown in the right corner of the corresponding figures. The photon transfer curves of the Flash2.8 sCMOS (e) and the iXon 897 EMCCD (f) were measured with the same camera settings as those in Fig. 3. The red arrows in (e) and (f) indicate the corresponding mean values of (a) and (b), which are originated from a combination of signal fluorescence and background fluorescence. The yellow arrows in (e) and (f) indicate the detected mean values of signal fluorescence, which was calculated from detected signal and SBR. The difference between the theoretical shot noise and total noise in (f) indicates the contribution of excess noise in EMCCD.

Tables (1)

Tables Icon

Table 1 Detectability of the Flash2.8 sCMOS and the iXon 897 EMCCD

Equations (24)

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I = I s i g × exp ( ( i x 0 ) 2 + ( j y 0 ) 2 2 s 2 ) + I b k g
< Δ x 2 > = s 2 + a 2 / 12 ϕ N + 8 π s 4 ( ϕ I b + N r 2 ) a 2 ( ϕ N ) 2
< Δ x 2 > = 2 s 2 + a 2 / 12 ϕ N + 8 π s 4 ( ϕ I b ) a 2 ( ϕ N ) 2
S N R = I s i g I s i g + σ I b k g 2
S B R = I s i g I b k g
σ Re a d , D N 2 = σ D i f f e r e n c e d F r a m e s , D N 2 = j = 1 99 i = 1 N p { [ S ( j ) i M ( j ) ] [ S ( j + 1 ) i M ( j + 1 ) ] } 2 2 × 99 × N p
S p a t i a l r e a d n o i s e = σ Re a d , D N
O F F i = j = 1 100 X ( j ) i 100
M ( j ) = i = 1 N p [ X ( j ) i O F F i ] N p
S ( j ) i = X ( j ) i O F F i
O F F = j = 1 100 i = 1 N p X ( j ) i 100 × N p
M ( j ) = i = 1 N p X ( j ) i N p O F F
S ( j ) i = X ( j ) i O F F
σ ( i ) Re a d , D N , t e m p o r a l 2 = j = 1 100 [ X ( j ) i M i ] 2 100
T e m p o r a l r e a d n o i s e = σ Re a d , D N , t e m p o r a l
σ T o t a l , D N 2 = j = 1 100 i = 1 N p [ S ( j ) i M ( j ) ] 2 100 × N p
σ S h o t , D N 2 + σ Re a d , D N 2 = σ D i f f e r e n c e d F r a m e s , D N 2 = j = 1 99 i = 1 N p { [ S ( j ) i M ( j ) ] [ S ( j + 1 ) i M ( j + 1 ) ] } 2 2 × 99 × N p
σ S h o t , D N 2 = σ D i f f e r e n c e d F r a m e s , D N 2 σ Re a d , D N 2
σ F P , D N 2 = σ T o t a l , D N 2 σ Re a d , D N 2 σ S h o t , D N 2
S i g n a l D N = j = 1 100 i = 1 N p S ( j ) i 100 × N p
K A D C ( e D N ) = S i g n a l D N σ S h o t , D N 2
T o t a l n o i s e = σ T o t a l , D N
S h o t n o i s e = σ S h o t , D N
F P n o i s e = σ F P , D N

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