Abstract

Modern sCMOS cameras are attractive for single molecule localization microscopy (SMLM) due to their high speed but suffer from pixel non-uniformities that can affect localization precision and accuracy. We present a simplified sCMOS non-uniform noise model that incorporates pixel specific read-noise, offset and sensitivity variation. Using this model we develop a new weighted least squared (WLS) fitting method designed to remove the effect of sCMOS pixel non-uniformities. Simulations with the sCMOS noise model, performed to test under which conditions sCMOS specific localization corrections are required, suggested that pixel specific offsets should always be removed. In many applications with thick biological samples photon fluxes are sufficiently high that corrections of read-noise and sensitivity correction may be neglected. When correction is required, e.g. during fast imaging in thin samples, our WLS fit procedure recovered the performance of an equivalent sensor with uniform pixel properties and the fit estimates also attained the Cramer-Rao lower bound. Experiments with sub-resolution beads and a DNA origami test sample confirmed the results of the simulations. The WLS localization procedure is fast to converge, compatible with 2D, 3D and multi-emitter localization and thus provides a computationally efficient sCMOS localization approach compatible with most SMLM modalities.

Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI.

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2016 (2)

M. Dai, R. Jungmann, and P. Yin, “Optical imaging of individual biomolecules in densely packed clusters,” Nat. Nanotechnol. 11(9), 798–807 (2016).
[Crossref] [PubMed]

L. Li, M. Li, Z. Zhang, and Z.-L. Huang, “Assessing low-light cameras with photon transfer curve method,” J. Innov. Opt. Health Sci. 9(3), 1630008 (2016).
[Crossref]

2014 (1)

H. Deschout, F. C. Zanacchi, M. Mlodzianoski, A. Diaspro, J. Bewersdorf, S. T. Hess, and K. Braeckmans, “Precisely and accurately localizing single emitters in fluorescence microscopy,” Nat. Methods 11(3), 253–266 (2014).
[Crossref] [PubMed]

2013 (2)

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt. Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

2012 (2)

S. Saurabh, S. Maji, and M. P. Bruchez, “Evaluation of sCMOS cameras for detection and localization of single Cy5 molecules,” Opt. Express 20(7), 7338–7349 (2012).
[Crossref] [PubMed]

T. J. Gould, S. T. Hess, and J. Bewersdorf, “Optical nanoscopy: from acquisition to analysis,” Annu. Rev. Biomed. Eng. 14(1), 231–254 (2012).
[Crossref] [PubMed]

2011 (3)

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

Z. L. Huang, H. Zhu, F. Long, H. Ma, L. Qin, Y. Liu, J. Ding, Z. Zhang, Q. Luo, and S. Zeng, “Localization-based super-resolution microscopy with an sCMOS camera,” Opt. Express 19(20), 19156–19168 (2011).
[Crossref] [PubMed]

2010 (2)

D. Baddeley, M. B. Cannell, and C. Soeller, “Visualization of localization microscopy data,” Microsc. Microanal. 16(1), 64–72 (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]

2008 (2)

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[Crossref] [PubMed]

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[Crossref] [PubMed]

2006 (3)

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. 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. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

2004 (1)

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

2000 (1)

M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

1994 (1)

Ahn, S.

S. Ahn and J. A. Fessler, “Standard errors of mean, variance, and standard deviation estimators,” (2003).

Baddeley, D.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

D. Baddeley, M. B. Cannell, and C. Soeller, “Visualization of localization microscopy data,” Microsc. Microanal. 16(1), 64–72 (2010).
[Crossref] [PubMed]

Baird, M. A.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Bates, M.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

Betzig, E.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[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]

Bewersdorf, J.

H. Deschout, F. C. Zanacchi, M. Mlodzianoski, A. Diaspro, J. Bewersdorf, S. T. Hess, and K. Braeckmans, “Precisely and accurately localizing single emitters in fluorescence microscopy,” Nat. Methods 11(3), 253–266 (2014).
[Crossref] [PubMed]

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

T. J. Gould, S. T. Hess, and J. Bewersdorf, “Optical nanoscopy: from acquisition to analysis,” Annu. Rev. Biomed. Eng. 14(1), 231–254 (2012).
[Crossref] [PubMed]

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]

Braeckmans, K.

H. Deschout, F. C. Zanacchi, M. Mlodzianoski, A. Diaspro, J. Bewersdorf, S. T. Hess, and K. Braeckmans, “Precisely and accurately localizing single emitters in fluorescence microscopy,” Nat. Methods 11(3), 253–266 (2014).
[Crossref] [PubMed]

Bruchez, M. P.

Cannell, M. B.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

D. Baddeley, M. B. Cannell, and C. Soeller, “Visualization of localization microscopy data,” Microsc. Microanal. 16(1), 64–72 (2010).
[Crossref] [PubMed]

Cheyne, J. E.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

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]

Cremer, C.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

Crossman, D.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

Dai, M.

M. Dai, R. Jungmann, and P. Yin, “Optical imaging of individual biomolecules in densely packed clusters,” Nat. Nanotechnol. 11(9), 798–807 (2016).
[Crossref] [PubMed]

Davidson, M. W.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[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]

Deschout, H.

H. Deschout, F. C. Zanacchi, M. Mlodzianoski, A. Diaspro, J. Bewersdorf, S. T. Hess, and K. Braeckmans, “Precisely and accurately localizing single emitters in fluorescence microscopy,” Nat. Methods 11(3), 253–266 (2014).
[Crossref] [PubMed]

Diaspro, A.

H. Deschout, F. C. Zanacchi, M. Mlodzianoski, A. Diaspro, J. Bewersdorf, S. T. Hess, and K. Braeckmans, “Precisely and accurately localizing single emitters in fluorescence microscopy,” Nat. Methods 11(3), 253–266 (2014).
[Crossref] [PubMed]

Ding, J.

Duim, W. C.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Fessler, J. A.

S. Ahn and J. A. Fessler, “Standard errors of mean, variance, and standard deviation estimators,” (2003).

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]

Galbraith, C. G.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[Crossref] [PubMed]

Galbraith, J. A.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[Crossref] [PubMed]

Girirajan, T. P.

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

Gould, T. J.

T. J. Gould, S. T. Hess, and J. Bewersdorf, “Optical nanoscopy: from acquisition to analysis,” Annu. Rev. Biomed. Eng. 14(1), 231–254 (2012).
[Crossref] [PubMed]

Gustafsson, M. G.

M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

Hartwich, T. M.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Heidbreder, M.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Heilemann, M.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Hell, S. W.

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.

H. Deschout, F. C. Zanacchi, M. Mlodzianoski, A. Diaspro, J. Bewersdorf, S. T. Hess, and K. Braeckmans, “Precisely and accurately localizing single emitters in fluorescence microscopy,” Nat. Methods 11(3), 253–266 (2014).
[Crossref] [PubMed]

T. J. Gould, S. T. Hess, and J. Bewersdorf, “Optical nanoscopy: from acquisition to analysis,” Annu. Rev. Biomed. Eng. 14(1), 231–254 (2012).
[Crossref] [PubMed]

S. T. Hess, T. P. 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.

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt. Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[Crossref] [PubMed]

Huang, F.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Huang, Z. L.

Huang, Z.-L.

L. Li, M. Li, Z. Zhang, and Z.-L. Huang, “Assessing low-light cameras with photon transfer curve method,” J. Innov. Opt. Health Sci. 9(3), 1630008 (2016).
[Crossref]

Jayasinghe, I. D.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

Jungmann, R.

M. Dai, R. Jungmann, and P. Yin, “Optical imaging of individual biomolecules in densely packed clusters,” Nat. Nanotechnol. 11(9), 798–807 (2016).
[Crossref] [PubMed]

Kamiyama, D.

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt. Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

Klein, T.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Li, L.

L. Li, M. Li, Z. Zhang, and Z.-L. Huang, “Assessing low-light cameras with photon transfer curve method,” J. Innov. Opt. Health Sci. 9(3), 1630008 (2016).
[Crossref]

Li, M.

L. Li, M. Li, Z. Zhang, and Z.-L. Huang, “Assessing low-light cameras with photon transfer curve method,” J. Innov. Opt. Health Sci. 9(3), 1630008 (2016).
[Crossref]

Lin, Y.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

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.

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, Y.

Long, F.

Long, J. J.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Löschberger, A.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Luo, Q.

Ma, H.

Maji, S.

Mason, M. D.

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

McGorty, R.

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt. Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

Mlodzianoski, M.

H. Deschout, F. C. Zanacchi, M. Mlodzianoski, A. Diaspro, J. Bewersdorf, S. T. Hess, and K. Braeckmans, “Precisely and accurately localizing single emitters in fluorescence microscopy,” Nat. Methods 11(3), 253–266 (2014).
[Crossref] [PubMed]

Montgomery, J. M.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

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]

Mothes, W.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Myers, J. R.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[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. 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]

Patterson, G. H.

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]

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]

Rivera-Molina, F. E.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Rossberger, S.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[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), 793–795 (2006).
[Crossref] [PubMed]

Sauer, M.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Saurabh, S.

Shroff, H.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[Crossref] [PubMed]

Soeller, C.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

D. Baddeley, M. B. Cannell, and C. Soeller, “Visualization of localization microscopy data,” Microsc. Microanal. 16(1), 64–72 (2010).
[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]

Toomre, D.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Uchil, P. D.

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

van de Linde, S.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Wang, W.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[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]

Wichmann, J.

Wolter, S.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Yin, P.

M. Dai, R. Jungmann, and P. Yin, “Optical imaging of individual biomolecules in densely packed clusters,” Nat. Nanotechnol. 11(9), 798–807 (2016).
[Crossref] [PubMed]

Zanacchi, F. C.

H. Deschout, F. C. Zanacchi, M. Mlodzianoski, A. Diaspro, J. Bewersdorf, S. T. Hess, and K. Braeckmans, “Precisely and accurately localizing single emitters in fluorescence microscopy,” Nat. Methods 11(3), 253–266 (2014).
[Crossref] [PubMed]

Zeng, S.

Zhang, Z.

L. Li, M. Li, Z. Zhang, and Z.-L. Huang, “Assessing low-light cameras with photon transfer curve method,” J. Innov. Opt. Health Sci. 9(3), 1630008 (2016).
[Crossref]

Z. L. Huang, H. Zhu, F. Long, H. Ma, L. Qin, Y. Liu, J. Ding, Z. Zhang, Q. Luo, and S. Zeng, “Localization-based super-resolution microscopy with an sCMOS camera,” Opt. Express 19(20), 19156–19168 (2011).
[Crossref] [PubMed]

Zhu, H.

Zhuang, X.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

Annu. Rev. Biomed. Eng. (1)

T. J. Gould, S. T. Hess, and J. Bewersdorf, “Optical nanoscopy: from acquisition to analysis,” Annu. Rev. Biomed. Eng. 14(1), 231–254 (2012).
[Crossref] [PubMed]

Biophys. J. (2)

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

J. Innov. Opt. Health Sci. (1)

L. Li, M. Li, Z. Zhang, and Z.-L. Huang, “Assessing low-light cameras with photon transfer curve method,” J. Innov. Opt. Health Sci. 9(3), 1630008 (2016).
[Crossref]

J. Microsc. (1)

M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[Crossref] [PubMed]

Microsc. Microanal. (1)

D. Baddeley, M. B. Cannell, and C. Soeller, “Visualization of localization microscopy data,” Microsc. Microanal. 16(1), 64–72 (2010).
[Crossref] [PubMed]

Nat. Methods (5)

H. Deschout, F. C. Zanacchi, M. Mlodzianoski, A. Diaspro, J. Bewersdorf, S. T. Hess, and K. Braeckmans, “Precisely and accurately localizing single emitters in fluorescence microscopy,” Nat. Methods 11(3), 253–266 (2014).
[Crossref] [PubMed]

F. Huang, T. M. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods 5(5), 417–423 (2008).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (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).
[Crossref] [PubMed]

Nat. Nanotechnol. (1)

M. Dai, R. Jungmann, and P. Yin, “Optical imaging of individual biomolecules in densely packed clusters,” Nat. Nanotechnol. 11(9), 798–807 (2016).
[Crossref] [PubMed]

Nat. Protoc. (1)

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Opt. Express (2)

Opt. Lett. (1)

Opt. Nanoscopy (1)

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt. Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

PLoS One (1)

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS One 6(5), e20645 (2011).
[Crossref] [PubMed]

Science (2)

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]

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[Crossref] [PubMed]

Other (2)

Hamamatsu, “Using Super-Resolution Nanorulers to study the Capabilities of EM-CCD and sCMOS Cameras beyond the Diffraction Limit,” (Hamamatsu Application Note).

S. Ahn and J. A. Fessler, “Standard errors of mean, variance, and standard deviation estimators,” (2003).

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Figures (9)

Fig. 1
Fig. 1

Maps and associated histograms of the (a) offset, (b) temporal variance and (c) flat-fielding values (sensitivity variation) of an Andor Zyla 4.2 sCMOS camera. The camera has a 2048x2048 pixel active area with a physical pixel size of 6.5 µm. The offset and variance were measured with a frame integration time of 50 ms. The offset ranges from 92 ADUs (Analog to Digital Units) to 879 ADUs with a mean of 103 ADUs and a standard deviation of 1.76 ADUs. The variance ranges from 1 to 867 e−2 (i.e. read noise 1 to ~30 e-) with a mean of 4 e−2. The flat-fielding value distribution has a mean of 1.0 and a standard deviation of ~0.4%.

Fig. 2
Fig. 2

Comparison of the error (measured as standard deviation of simulation errors) of our WLS algorithm and the CRLB for the error in the x coordinate as a function of event photon count at a range of background levels. CRLB = Cramer-Rao lower bound; WLS = weighted least-squares; bg = background level (e-).

Fig. 3
Fig. 3

Simulation of read noise effects on emitter localization. (a) A pixel with configurable read noise is located ~2 pixels to the left of the center of a 1K photons/event single emitter. (b) With low background, a read noise of 20 e- introduces a broadening effect using ‘uncorrected localization’. Using ‘corrected localization’, the localization precision is recovered. (c) A schematic comparison of localization with map correction (blue), the uncorrected algorithm (green) and for comparison simulated uniform sensor data localized with the standard algorithm (dashed). Centers of ellipses represent the mean positions of the localization clouds (localization bias), and the major/minor axes show the localization precision. (d) Effect of a noisy pixel on localization precision as a function of background level. (e) Map of localization precision versus read noise and background, event photon count = 1K photons. (f) Map of localization precision versus read noise and event photon count at a fixed background level of 50 photons. (g) Map of localization bias versus read noise and background, 1K photon events. (e)-(g) were calculated using the uncorrected algorithm. loc err = localization error.

Fig. 4
Fig. 4

Simulation of the effect of uncorrected sensitivity variation on the localization of a single emitter. (a) Simulated frames for different values of sensitivity variation obtained by scaling of our measured flatfield map. The emitter has a photon count of 2000 with a background of 200 e-. A clear vertical stripe pattern is observed when the simulated sensitivity variation is ≥5%. (b) Localization results with different sensitivity variation levels. The centers of ellipses represent the mean positions of the localization clouds, i.e. localization bias, and the major/minor axes represent the localization precisions. (c) 3D plot of the ratio of bias and precision for different sensitivity variation levels, event photon counts and background levels. Abbreviation: loc err = localization error; sv = sensitivity variation.

Fig. 5
Fig. 5

Simulation of hot pixel effects on emitter localization. (a) Plot of temporal variance vs dark signal of pixels with offset >150 ADUs. The dashed line is a line of equal variance to dark signal. (b) A simulated frame of a emitter (1k photons) with a background of 100 e- and a hot pixel of 400 ADUs. (c) A bias of ~18 nm is introduced into the localization of the emitter in (b). (d) The bias in (c) is eliminated by using either blemish correction (blue) or standard map correction (cyan), both achieve localization equivalent to a uniform sensor (dashed red). The crosses represent the mean localization of the emitter and the axes represent localization precision. (e) Bias from hot pixel presence and backgrounds, event photon count is 1000 photons. (f) Bias from hot pixel presence and event photon counts. Background = 200 e-.

Fig. 6
Fig. 6

Simulation of localization with different spatial sampling rates. (a) Localization precision versus pixel sampling for 1000 photon emitter events and 50 e- background/104nm2. (b) Corresponding localization bias versus pixel sampling. Uniform sensor data is shown as reference versus an sCMOS model with a high read noise pixel (22 e-) ~2 pixels from the center, using the WLS algorithm with and without map correction. The PSF model had a FWHM diameter of 230 nm. scmos uncorr/corr = scmos localization without/with correction.

Fig. 7
Fig. 7

Simulation of 3D localization in sCMOS data. (a) PSF at z-steps of 50nm, photon event count = 1000, background = 10 e-. (b) and (c) Estimation of the axial position and error without algorithmic correction. The z-position of the emitter is varied over a range of 800 nm around the focal plane. (d) and (e) Estimation of the axial position and error with map based correction. An improvement of axial localization precision from 60.0nm to 27.7nm and bias from 17.8nm to 0.3nm is achieved. (f) Simulated frames at different emitter positions. (g) A schematic comparison of the sCMOS and standard algorithm. Blue: sCMOS algorithm; green: standard algorithm; dashed: localizations from an equivalent uniform sensor.

Fig. 8
Fig. 8

Application of the sCMOS algorithm and effect of read noise and background. (a) Temporal variance image of a 3K-frame image series reveals a noisy pixel (~15 e-) close to a bead (brightness ~1800 photons). (b) A single frame of the image series with low background (5 e-). (c), (e) and (g) Localizations of the bead in (b) using the standard algorithm. (d), (f) and (h) Localizations of the bead in (b) using the sCMOS algorithm. (i) A single frame of the image series with high background (90 e-). (j), (l) and (n) Localizations of the bead in (i) using the standard algorithm. (k), (m) and (o) Localizations of the bead in (i) using the sCMOS algorithm.

Fig. 9
Fig. 9

Imaging of a nanoruler sample with corrected and uncorrected localization algorithms. (a) Image reconstructed using the sCMOS corrected WLS algorithm, and (b) using the same raw frame data but processed with the uncorrected algorithm. The area in the green box is compared in detail in (c), (d) and also related to an image of the same structure obtained with a more uniform sensor, an Andor Ixon EMCCD (e). (f) and (g). Comparison of intensity profiles through the structure as shown in (c) and (e). (h). Corresponding localization errors estimated from the co-variance of the WLS penalty function at convergence. (i) Distribution of localizations along the direction orthogonal to the origami axis in (c) and (e).

Equations (10)

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

I raw = 1 N 1 N I i o
I filtered =gaussian_filter( I raw )
d i = g av f i ( d raw,i o i )
ε i 2 = ε RN,i 2 + s i 2
E(λ)= 0 λp(λ| d i )dλ
P(λ|d ) i = p( d i |λ)p(λ) p( d i )
E(λ)= 0 λp( λ| d i )dλ 0 λp( d i |λ)dλ= 0 λ d i +1 e λ d i ! dλ
E(λ)= Γ( d i +2) d i ! = ( d i +1)! d i ! = d i +1
ε i 2 = ε RN,i 2 +max( d i ,0)+1
χ 2 = i w i ( d i E model,i ( x c , y c , z c ,A,σ, b 2 )) 2

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