Abstract

Localization of single molecules in microscopy images is a key step in quantitative single particle data analysis. Among them, single molecule based super-resolution optical microscopy techniques require high localization accuracy as well as computation of large data sets in the order of 105 single molecule detections to reconstruct a single image. We hereby present an algorithm based on image wavelet segmentation and single particle centroid determination, and compare its performance with the commonly used Gaussian fitting of the point spread function. We performed realistic simulations at different signal-to-noise ratios and particle densities and show that the calculation time using the wavelet approach can be more than one order of magnitude faster than that of Gaussian fitting without a significant degradation of the localization accuracy, from 1 nm to 4 nm in our range of study. We propose a simulation-based estimate of the resolution of an experimental single molecule acquisition.

© 2012 OSA

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2011 (6)

S. J. Holden, S. Uphoff, and A. N. Kapanidis, “DAOSTORM: an algorithm for high- density super-resolution microscopy,” Nat. Methods 8(4), 279–280 (2011).
[CrossRef] [PubMed]

I. Izeddin, C. G. Specht, M. Lelek, X. Darzacq, A. Triller, C. Zimmer, and M. Dahan, “Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe,” PLoS ONE 6(1), e15611 (2011).
[CrossRef] [PubMed]

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]

P. Křížek, I. Raška, and G. M. Hagen, “Minimizing detection errors in single molecule localization microscopy,” Opt. Express 19(4), 3226–3235 (2011).
[CrossRef] [PubMed]

S. Wolter, U. Endesfelder, S. van de Linde, M. Heilemann, and M. Sauer, “Measuring localization performance of super-resolution algorithms on very active samples,” Opt. Express 19(8), 7020–7033 (2011).
[CrossRef] [PubMed]

F. Huang, S. L. Schwartz, J. M. Byars, and K. A. Lidke, “Simultaneous multiple-emitter fitting for single molecule super-resolution imaging,” Biomed. Opt. Express 2(5), 1377–1393 (2011).
[CrossRef] [PubMed]

2010 (4)

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

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]

R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7(5), 339–340 (2010).
[CrossRef] [PubMed]

S. J. Lord, H.-L. D. Lee, and W. E. Moerner, “Single-molecule spectroscopy and imaging of biomolecules in living cells,” Anal. Chem. 82(6), 2192–2203 (2010).
[CrossRef] [PubMed]

2009 (4)

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

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]

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

A. V. Abraham, S. Ram, J. Chao, E. S. Ward, and R. J. Ober, “Quantitative study of single molecule location estimation techniques,” Opt. Express 17(26), 23352–23373 (2009).
[CrossRef] [PubMed]

2008 (4)

S. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5(2), 155–157 (2008).
[CrossRef] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, “Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes,” Nat. Methods 5(8), 687–694 (2008).
[CrossRef] [PubMed]

A. Triller and D. Choquet, “New concepts in synaptic biology derived from single-molecule imaging,” Neuron 59(3), 359–374 (2008).
[CrossRef] [PubMed]

2007 (1)

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

2003 (2)

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science 300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

M. Dahan, S. Lévi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, “Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking,” Science 302(5644), 442–445 (2003).
[CrossRef] [PubMed]

2002 (2)

J. Starck, E. Pantin, and F. Murtagh, “Deconvolution in astronomy: A review,” Publ. Astron. Soc. Pac. 114(800), 1051–1069 (2002).
[CrossRef]

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

2001 (1)

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 (1)

U. Kubitscheck, O. Kückmann, T. Kues, and R. Peters, “Imaging and tracking of single GFP molecules in solution,” Biophys. J. 78(4), 2170–2179 (2000).
[CrossRef] [PubMed]

1995 (1)

D. Donoho and I. Johnstone, “Adapting to unknown smoothness via wavelet shrinkage,” J. Am. Stat. Assoc. 90(432), 1200–1224 (1995).
[CrossRef]

1991 (1)

L. Vincent and P. Soille, “Watersheds in digital spaces - an efficient algorithm based on immersion simulations,” IEEE Trans. Pattern Anal. Mach. Intell. 13(6), 583–598 (1991).
[CrossRef]

Abraham, A. V.

Bates, M.

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

Bertaux, N.

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, “Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes,” Nat. Methods 5(8), 687–694 (2008).
[CrossRef] [PubMed]

Betzig, E.

S. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5(2), 155–157 (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]

Bock, H.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[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]

Bossi, M.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Byars, J. M.

Chao, J.

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]

Choquet, D.

A. Triller and D. Choquet, “New concepts in synaptic biology derived from single-molecule imaging,” Neuron 59(3), 359–374 (2008).
[CrossRef] [PubMed]

Dahan, M.

I. Izeddin, C. G. Specht, M. Lelek, X. Darzacq, A. Triller, C. Zimmer, and M. Dahan, “Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe,” PLoS ONE 6(1), e15611 (2011).
[CrossRef] [PubMed]

M. Dahan, S. Lévi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, “Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking,” Science 302(5644), 442–445 (2003).
[CrossRef] [PubMed]

Darzacq, X.

I. Izeddin, C. G. Specht, M. Lelek, X. Darzacq, A. Triller, C. Zimmer, and M. Dahan, “Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe,” PLoS ONE 6(1), e15611 (2011).
[CrossRef] [PubMed]

Davidson, M.

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (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]

Donoho, D.

D. Donoho and I. Johnstone, “Adapting to unknown smoothness via wavelet shrinkage,” J. Am. Stat. Assoc. 90(432), 1200–1224 (1995).
[CrossRef]

Eggeling, C.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Endesfelder, U.

Fölling, J.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Forkey, J. N.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science 300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Fornasiero, E. F.

R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7(5), 339–340 (2010).
[CrossRef] [PubMed]

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]

Gillette, J. M.

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

S. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5(2), 155–157 (2008).
[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]

Goldman, Y. E.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science 300(5628), 2061–2065 (2003).
[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]

Ha, T.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science 300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Hagen, G. M.

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]

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]

S. Wolter, U. Endesfelder, S. van de Linde, M. Heilemann, and M. Sauer, “Measuring localization performance of super-resolution algorithms on very active samples,” Opt. Express 19(8), 7020–7033 (2011).
[CrossRef] [PubMed]

Hein, B.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Hell, S. W.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Henriques, R.

R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7(5), 339–340 (2010).
[CrossRef] [PubMed]

Hess, H. F.

S. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5(2), 155–157 (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]

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]

Holden, S. J.

S. J. Holden, S. Uphoff, and A. N. Kapanidis, “DAOSTORM: an algorithm for high- density super-resolution microscopy,” Nat. Methods 8(4), 279–280 (2011).
[CrossRef] [PubMed]

Huang, B.

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

Huang, F.

Izeddin, I.

I. Izeddin, C. G. Specht, M. Lelek, X. Darzacq, A. Triller, C. Zimmer, and M. Dahan, “Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe,” PLoS ONE 6(1), e15611 (2011).
[CrossRef] [PubMed]

Jakobs, S.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Johnstone, I.

D. Donoho and I. Johnstone, “Adapting to unknown smoothness via wavelet shrinkage,” J. Am. Stat. Assoc. 90(432), 1200–1224 (1995).
[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.

S. J. Holden, S. Uphoff, and A. N. Kapanidis, “DAOSTORM: an algorithm for high- density super-resolution microscopy,” Nat. Methods 8(4), 279–280 (2011).
[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]

Krížek, P.

Kubitscheck, U.

U. Kubitscheck, O. Kückmann, T. Kues, and R. Peters, “Imaging and tracking of single GFP molecules in solution,” Biophys. J. 78(4), 2170–2179 (2000).
[CrossRef] [PubMed]

Kückmann, O.

U. Kubitscheck, O. Kückmann, T. Kues, and R. Peters, “Imaging and tracking of single GFP molecules in solution,” Biophys. J. 78(4), 2170–2179 (2000).
[CrossRef] [PubMed]

Kues, T.

U. Kubitscheck, O. Kückmann, T. Kues, and R. Peters, “Imaging and tracking of single GFP molecules in solution,” Biophys. J. 78(4), 2170–2179 (2000).
[CrossRef] [PubMed]

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

Lee, H.-L. D.

S. J. Lord, H.-L. D. Lee, and W. E. Moerner, “Single-molecule spectroscopy and imaging of biomolecules in living cells,” Anal. Chem. 82(6), 2192–2203 (2010).
[CrossRef] [PubMed]

Lelek, M.

I. Izeddin, C. G. Specht, M. Lelek, X. Darzacq, A. Triller, C. Zimmer, and M. Dahan, “Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe,” PLoS ONE 6(1), e15611 (2011).
[CrossRef] [PubMed]

R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7(5), 339–340 (2010).
[CrossRef] [PubMed]

Lévi, S.

M. Dahan, S. Lévi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, “Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking,” Science 302(5644), 442–445 (2003).
[CrossRef] [PubMed]

Lidke, K. A.

F. Huang, S. L. Schwartz, J. M. Byars, and K. A. Lidke, “Simultaneous multiple-emitter fitting for single molecule super-resolution imaging,” Biomed. Opt. Express 2(5), 1377–1393 (2011).
[CrossRef] [PubMed]

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. 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.

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

S. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5(2), 155–157 (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]

Lord, S. J.

S. J. Lord, H.-L. D. Lee, and W. E. Moerner, “Single-molecule spectroscopy and imaging of biomolecules in living cells,” Anal. Chem. 82(6), 2192–2203 (2010).
[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]

Luccardini, C.

M. Dahan, S. Lévi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, “Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking,” Science 302(5644), 442–445 (2003).
[CrossRef] [PubMed]

Manley, S.

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

S. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5(2), 155–157 (2008).
[CrossRef] [PubMed]

Marguet, D.

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, “Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes,” Nat. Methods 5(8), 687–694 (2008).
[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]

McKinney, S. A.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science 300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Medda, R.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Mhlanga, M. M.

R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7(5), 339–340 (2010).
[CrossRef] [PubMed]

Moerner, W. E.

S. J. Lord, H.-L. D. Lee, and W. E. Moerner, “Single-molecule spectroscopy and imaging of biomolecules in living cells,” Anal. Chem. 82(6), 2192–2203 (2010).
[CrossRef] [PubMed]

Murtagh, F.

J. Starck, E. Pantin, and F. Murtagh, “Deconvolution in astronomy: A review,” Publ. Astron. Soc. Pac. 114(800), 1051–1069 (2002).
[CrossRef]

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]

Ober, R. J.

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]

Olivo-Marin, J.-C.

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]

Pantin, E.

J. Starck, E. Pantin, and F. Murtagh, “Deconvolution in astronomy: A review,” Publ. Astron. Soc. Pac. 114(800), 1051–1069 (2002).
[CrossRef]

Patterson, G.

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

Patterson, G. H.

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

S. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5(2), 155–157 (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]

Peters, R.

U. Kubitscheck, O. Kückmann, T. Kues, and R. Peters, “Imaging and tracking of single GFP molecules in solution,” Biophys. J. 78(4), 2170–2179 (2000).
[CrossRef] [PubMed]

Ram, S.

Raška, I.

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]

Rigneault, H.

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, “Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes,” Nat. Methods 5(8), 687–694 (2008).
[CrossRef] [PubMed]

Riveau, B.

M. Dahan, S. Lévi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, “Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking,” Science 302(5644), 442–445 (2003).
[CrossRef] [PubMed]

Rostaing, P.

M. Dahan, S. Lévi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, “Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking,” Science 302(5644), 442–445 (2003).
[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]

Sauer, M.

S. Wolter, U. Endesfelder, S. van de Linde, M. Heilemann, and M. Sauer, “Measuring localization performance of super-resolution algorithms on very active samples,” Opt. Express 19(8), 7020–7033 (2011).
[CrossRef] [PubMed]

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]

Schwartz, S. L.

Selvin, P. R.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science 300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Sergé, A.

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, “Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes,” Nat. Methods 5(8), 687–694 (2008).
[CrossRef] [PubMed]

Shroff, H.

S. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5(2), 155–157 (2008).
[CrossRef] [PubMed]

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).
[CrossRef] [PubMed]

Soille, P.

L. Vincent and P. Soille, “Watersheds in digital spaces - an efficient algorithm based on immersion simulations,” IEEE Trans. Pattern Anal. Mach. Intell. 13(6), 583–598 (1991).
[CrossRef]

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]

Specht, C. G.

I. Izeddin, C. G. Specht, M. Lelek, X. Darzacq, A. Triller, C. Zimmer, and M. Dahan, “Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe,” PLoS ONE 6(1), e15611 (2011).
[CrossRef] [PubMed]

Starck, J.

J. Starck, E. Pantin, and F. Murtagh, “Deconvolution in astronomy: A review,” Publ. Astron. Soc. Pac. 114(800), 1051–1069 (2002).
[CrossRef]

Subach, F. V.

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

Triller, A.

I. Izeddin, C. G. Specht, M. Lelek, X. Darzacq, A. Triller, C. Zimmer, and M. Dahan, “Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe,” PLoS ONE 6(1), e15611 (2011).
[CrossRef] [PubMed]

A. Triller and D. Choquet, “New concepts in synaptic biology derived from single-molecule imaging,” Neuron 59(3), 359–374 (2008).
[CrossRef] [PubMed]

M. Dahan, S. Lévi, C. Luccardini, P. Rostaing, B. Riveau, and A. Triller, “Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking,” Science 302(5644), 442–445 (2003).
[CrossRef] [PubMed]

Uphoff, S.

S. J. Holden, S. Uphoff, and A. N. Kapanidis, “DAOSTORM: an algorithm for high- density super-resolution microscopy,” Nat. Methods 8(4), 279–280 (2011).
[CrossRef] [PubMed]

Valtorta, F.

R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7(5), 339–340 (2010).
[CrossRef] [PubMed]

van de Linde, S.

S. Wolter, U. Endesfelder, S. van de Linde, M. Heilemann, and M. Sauer, “Measuring localization performance of super-resolution algorithms on very active samples,” Opt. Express 19(8), 7020–7033 (2011).
[CrossRef] [PubMed]

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]

Verkhusha, V. V.

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

Vincent, L.

L. Vincent and P. Soille, “Watersheds in digital spaces - an efficient algorithm based on immersion simulations,” IEEE Trans. Pattern Anal. Mach. Intell. 13(6), 583–598 (1991).
[CrossRef]

Walker, W. F.

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.

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[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]

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]

S. Wolter, U. Endesfelder, S. van de Linde, M. Heilemann, and M. Sauer, “Measuring localization performance of super-resolution algorithms on very active samples,” Opt. Express 19(8), 7020–7033 (2011).
[CrossRef] [PubMed]

Wurm, C. A.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[CrossRef] [PubMed]

Yildiz, A.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science 300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Zerubia, J.

Zhang, B.

Zhuang, X.

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

Zimmer, C.

I. Izeddin, C. G. Specht, M. Lelek, X. Darzacq, A. Triller, C. Zimmer, and M. Dahan, “Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe,” PLoS ONE 6(1), e15611 (2011).
[CrossRef] [PubMed]

R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7(5), 339–340 (2010).
[CrossRef] [PubMed]

Anal. Chem. (1)

S. J. Lord, H.-L. D. Lee, and W. E. Moerner, “Single-molecule spectroscopy and imaging of biomolecules in living cells,” Anal. Chem. 82(6), 2192–2203 (2010).
[CrossRef] [PubMed]

Annu. Rev. Biochem. (1)

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

Annu. Rev. Phys. Chem. (1)

G. Patterson, M. Davidson, S. Manley, and J. Lippincott-Schwartz, “Superresolution imaging using single-molecule localization,” Annu. Rev. Phys. Chem. 61(1), 345–367 (2010).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biomed. Opt. Express (1)

Biophys. J. (4)

U. Kubitscheck, O. Kückmann, T. Kues, and R. Peters, “Imaging and tracking of single GFP molecules in solution,” Biophys. J. 78(4), 2170–2179 (2000).
[CrossRef] [PubMed]

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[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. 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]

IEEE Trans. Pattern Anal. Mach. Intell. (1)

L. Vincent and P. Soille, “Watersheds in digital spaces - an efficient algorithm based on immersion simulations,” IEEE Trans. Pattern Anal. Mach. Intell. 13(6), 583–598 (1991).
[CrossRef]

J. Am. Stat. Assoc. (1)

D. Donoho and I. Johnstone, “Adapting to unknown smoothness via wavelet shrinkage,” J. Am. Stat. Assoc. 90(432), 1200–1224 (1995).
[CrossRef]

Nat. Methods (9)

S. J. Holden, S. Uphoff, and A. N. Kapanidis, “DAOSTORM: an algorithm for high- density super-resolution microscopy,” Nat. Methods 8(4), 279–280 (2011).
[CrossRef] [PubMed]

F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz, and V. V. Verkhusha, “Photoactivatable mCherry for high-resolution two-color fluorescence microscopy,” Nat. Methods 6(2), 153–159 (2009).
[CrossRef] [PubMed]

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, “Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes,” Nat. Methods 5(8), 687–694 (2008).
[CrossRef] [PubMed]

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]

R. Henriques, M. Lelek, E. F. Fornasiero, F. Valtorta, C. Zimmer, and M. M. Mhlanga, “QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ,” Nat. Methods 7(5), 339–340 (2010).
[CrossRef] [PubMed]

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. Manley, J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz, “High-density mapping of single-molecule trajectories with photoactivated localization microscopy,” Nat. Methods 5(2), 155–157 (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), 793–796 (2006).
[CrossRef] [PubMed]

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Figures (7)

Fig. 1
Fig. 1

Simulations of single particles. Examples of the simulated single molecule data on a 256 px x 256 px matrix of 100 nm pixel size. Each single point is convolved with a 2D Gaussian function with variance σ2 = 1 px, and the image corrupted with a mixture of Gaussian and Poisson noise. In panel A, examples at SNR = 7.1 and molecule density of 0.1, 0.25, 0.5, 0.75, and 1 molecule/μm2 (left to right). In panel B, examples at molecular density of 0.1 molecule/μm2 and SNR = 10.3, 6.6, 3.6, 3, and 2.6 (left to right). In panel C, intensity profile across one single molecule for different SNR, normalized in the inset, and shown as raw images on the right.

Fig. 2
Fig. 2

Wavelet segmentation. From left to right: 1) source images with the localization of single molecules in green areas; 2) 1st wavelet plane; 3) 2nd wavelet plane; 4) segmented images; 5) centroid computation for each localized molecule defined by the segmented image. Red and orange markers (*) illustrate false position determination and false negative detection, respectively. From top to bottom: illustrations with different signal-to-noise ratio (10.3, 3.6, and 3, respectively), using the same segmentation threshold defined by 0.5 time the standard deviation of the noisiest image.

Fig. 3
Fig. 3

Computation time. In panels A and B, comparisons of the computation time between the wavelet segmentation and the MTT algorithms. The total time needed to detect and determine the position of a series of 100 images with simulated single molecules, are represented as a function of the molecular density for different SNRs (A), and as a function of SNR for several molecular densities (B). Panel C shows a comparison between the wavelet algorithm and QuickPALM for a series of 4000 images at different molecular density. With QuickPALM, two to four CPU processors were used for the computation.

Fig. 4
Fig. 4

Localization accuracy. Comparison of the localization accuracy between the wavelet segmentation and the MTT algorithms. The localization accuracy was calculated as mean value of the positioning error calculated for all the detections of each data set, and is shown as a function of the SNR, for several molecular densities.

Fig. 5
Fig. 5

False detections. Rates of false positive and false negative detections, in a radius of 100 nm around the coordinates of each simulated single molecule. In panel A, comparison of the false positive rate between the wavelet approach and MTT, as a function of the SNR, for several molecular densities. In panel B, false negative rates as a function of the SNR.

Fig. 6
Fig. 6

Simulation of test patters. Examples of simulated single molecule data inside a test pattern made of alternating stripes of variable width ranging from 200 nm (periphery) down to 6 nm (center). Panel A: the simulated pattern, ideal (top) and reconstructed from a limited number of molecules (bottom). Panel B: reconstructed images after localization for various SNRs and molecule densities. Panel C: contrast functions and their corresponding modulation contrast functions.

Fig. 7
Fig. 7

Experimental data. Performance of the wavelet segmentation algorithm and the MTT algorithm with experimental PALM data. Panel A: Low-resolution image (left) and super-resolution reconstructions (center and right) of the actin cytoskeleton of rat hippocampal neurons expressing ABP-tdEosFP and fixed with 4% paraformaldehyde. 50 000 individual frames were analyzed; the analysis with the wavelet segmentation algorithm took 7.7 minutes and identified 1 058 886 single molecule events, while the Gaussian approach took 116 minutes and detected 1 092 266 events. Panel B: Comparison of the intensity profiles across the shaft of the dendrite for the images shown in A (indicated by a white line). Panel C: higher magnification of a 2.65 x 2.65 µm2 region (boxed region in A) containing an individual dendritic spine where 45 878 single molecule events were detected with the wavelet segmentation algorithm (top panel, detected particles are overlaid with the diffraction limited image of the pre-converted fluorophores); a super-resolution reconstruction is shown with a pixel size of 9.6 nm with each single molecule represented by one pixel of unitary intensity (lower panel). Panel D: Histogram of the SNR values of the single fluorophore intensities detected in the region, with a mean SNR of 5.3, and the mean values of the SNR considering the brightest 10%, 25%, 50%, 75%, and 100% detections.

Tables (2)

Tables Icon

Table 1 Steps followed by the Gaussian and wavelet approaches for particle detection and position determination.

Tables Icon

Table 2 Summary of the localization accuracy achieved with the wavelet algorithm approach for the sets of simulated data at different particle concentrations and SNRs. In brackets, localization accuracy as determined by the cutoff of the abscissa for the fit of the data points of the modulation transfer function above 15% normalized intensity.

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