Abstract

We propose a method for simultaneously measuring the position and emission color of single fluorescent emitters based on the use of a large pitch diffraction grating in the emission light path. The grating produces satellite spots adjacent to the main spot; the relative distance between the spots is a measure for the emission wavelength. We present proof-of-principle experiments on beads and mixtures of quantum dots using a spatial light modulator for making a programmable diffraction grating. A wavelength precision of around 10 nm can be achieved for 1000 signal photons and practical background levels, while maintaining a localization precision of around 10 nm.

© 2014 Optical Society of America

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  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, Science 313, 1642 (2006).
    [CrossRef]
  2. M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
    [CrossRef]
  3. S. T. Hess, T. P. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
    [CrossRef]
  4. H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
    [CrossRef]
  5. M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
    [CrossRef]
  6. I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
    [CrossRef]
  7. M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, Science 317, 1749 (2007).
    [CrossRef]
  8. P. J. Cutler, M. D. Malik, S. Liu, J. M. Byars, D. S. Lidke, and K. A. Lidke, PLoS One 8, e64320, 2013.
    [CrossRef]
  9. Y. Ma, M. R. Shortreed, and E. S. Yeung, Anal. Chem. 72, 4640 (2000).
    [CrossRef]
  10. G. A. Blab, S. Oelllerich, R. Schumm, and T. Schmidt, Opt. Lett. 29, 727 (2004).
    [CrossRef]
  11. K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, Nat. Methods 7, 377 (2010).
    [CrossRef]
  12. B. Rieger and S. Stallinga, Chem. Phys. Chem. 15, 664 (2014).
    [CrossRef]
  13. S. Stallinga and B. Rieger, Opt. Express 18, 24461 (2010).
    [CrossRef]
  14. A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, Nat. Methods 5, 687 (2008).
    [CrossRef]
  15. D. W. Scott, Biometrika 66, 605 (1979).
    [CrossRef]
  16. M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
    [CrossRef]
  17. D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, PloS One 6, e20645 (2011).
    [CrossRef]

2014 (1)

B. Rieger and S. Stallinga, Chem. Phys. Chem. 15, 664 (2014).
[CrossRef]

2013 (1)

P. J. Cutler, M. D. Malik, S. Liu, J. M. Byars, D. S. Lidke, and K. A. Lidke, PLoS One 8, e64320, 2013.
[CrossRef]

2011 (2)

M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
[CrossRef]

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, PloS One 6, e20645 (2011).
[CrossRef]

2010 (3)

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, Nat. Methods 7, 377 (2010).
[CrossRef]

S. Stallinga and B. Rieger, Opt. Express 18, 24461 (2010).
[CrossRef]

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

2008 (2)

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, Nat. Methods 5, 687 (2008).
[CrossRef]

2007 (2)

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
[CrossRef]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, Science 317, 1749 (2007).
[CrossRef]

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, Science 313, 1642 (2006).
[CrossRef]

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

S. T. Hess, T. P. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
[CrossRef]

2004 (1)

2000 (1)

Y. Ma, M. R. Shortreed, and E. S. Yeung, Anal. Chem. 72, 4640 (2000).
[CrossRef]

1979 (1)

D. W. Scott, Biometrika 66, 605 (1979).
[CrossRef]

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, PloS One 6, e20645 (2011).
[CrossRef]

Bates, M.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, Science 317, 1749 (2007).
[CrossRef]

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Belov, V. N.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

Bertaux, N.

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, Nat. Methods 5, 687 (2008).
[CrossRef]

Betzig, E.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
[CrossRef]

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, Science 313, 1642 (2006).
[CrossRef]

Blab, G. A.

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, Science 313, 1642 (2006).
[CrossRef]

Bossi, M.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

Boyarskiy, V. P.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

Byars, J. M.

P. J. Cutler, M. D. Malik, S. Liu, J. M. Byars, D. S. Lidke, and K. A. Lidke, PLoS One 8, e64320, 2013.
[CrossRef]

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, PloS One 6, e20645 (2011).
[CrossRef]

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, PloS One 6, e20645 (2011).
[CrossRef]

Churchman, L. S.

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, Nat. Methods 7, 377 (2010).
[CrossRef]

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, PloS One 6, e20645 (2011).
[CrossRef]

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, PloS One 6, e20645 (2011).
[CrossRef]

Cutler, P. J.

P. J. Cutler, M. D. Malik, S. Liu, J. M. Byars, D. S. Lidke, and K. A. Lidke, PLoS One 8, e64320, 2013.
[CrossRef]

Davidson, M. W.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
[CrossRef]

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, Science 313, 1642 (2006).
[CrossRef]

Dempsey, G. T.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, Science 317, 1749 (2007).
[CrossRef]

Eggeling, C.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

Egner, A.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

Flyvbjerg, H.

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, Nat. Methods 7, 377 (2010).
[CrossRef]

Folling, J.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

Fölling, J.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

Galbraith, C. G.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
[CrossRef]

Galbraith, J. A.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
[CrossRef]

Gillette, J.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
[CrossRef]

Girirajan, T. P.

S. T. Hess, T. P. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
[CrossRef]

Gould, T. J.

M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
[CrossRef]

Gudheti, M. V.

M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
[CrossRef]

Gunewardene, M. S.

M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
[CrossRef]

Hell, S. W.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

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, Science 313, 1642 (2006).
[CrossRef]

Hess, S. T.

M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
[CrossRef]

S. T. Hess, T. P. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
[CrossRef]

Huang, B.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, Science 317, 1749 (2007).
[CrossRef]

Jakobs, S.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[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, PloS One 6, e20645 (2011).
[CrossRef]

Lidke, D. S.

P. J. Cutler, M. D. Malik, S. Liu, J. M. Byars, D. S. Lidke, and K. A. Lidke, PLoS One 8, e64320, 2013.
[CrossRef]

Lidke, K. A.

P. J. Cutler, M. D. Malik, S. Liu, J. M. Byars, D. S. Lidke, and K. A. Lidke, PLoS One 8, e64320, 2013.
[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, Science 313, 1642 (2006).
[CrossRef]

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, Science 313, 1642 (2006).
[CrossRef]

Liu, S.

P. J. Cutler, M. D. Malik, S. Liu, J. M. Byars, D. S. Lidke, and K. A. Lidke, PLoS One 8, e64320, 2013.
[CrossRef]

Ma, Y.

Y. Ma, M. R. Shortreed, and E. S. Yeung, Anal. Chem. 72, 4640 (2000).
[CrossRef]

Malik, M. D.

P. J. Cutler, M. D. Malik, S. Liu, J. M. Byars, D. S. Lidke, and K. A. Lidke, PLoS One 8, e64320, 2013.
[CrossRef]

Marguet, D.

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, Nat. Methods 5, 687 (2008).
[CrossRef]

Mason, M. D.

S. T. Hess, T. P. Girirajan, and M. D. Mason, Biophys. J. 91, 4258 (2006).
[CrossRef]

Medda, R.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

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, PloS One 6, e20645 (2011).
[CrossRef]

Mortensen, K. I.

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, Nat. Methods 7, 377 (2010).
[CrossRef]

Oelllerich, S.

Olenych, S.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
[CrossRef]

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, Science 313, 1642 (2006).
[CrossRef]

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, Science 313, 1642 (2006).
[CrossRef]

Penoncello, G. P.

M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
[CrossRef]

Rieger, B.

B. Rieger and S. Stallinga, Chem. Phys. Chem. 15, 664 (2014).
[CrossRef]

S. Stallinga and B. Rieger, Opt. Express 18, 24461 (2010).
[CrossRef]

Rigneault, H.

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, Nat. Methods 5, 687 (2008).
[CrossRef]

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, PloS One 6, e20645 (2011).
[CrossRef]

Rothermel, E.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Schmidt, T.

Schonle, A.

M. Bossi, J. Folling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schonle, and S. W. Hell, Nano Lett. 8, 2463 (2008).
[CrossRef]

Schönle, A.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

Schumm, R.

Scott, D. W.

D. W. Scott, Biometrika 66, 605 (1979).
[CrossRef]

Sergé, A.

A. Sergé, N. Bertaux, H. Rigneault, and D. Marguet, Nat. Methods 5, 687 (2008).
[CrossRef]

Shortreed, M. R.

Y. Ma, M. R. Shortreed, and E. S. Yeung, Anal. Chem. 72, 4640 (2000).
[CrossRef]

Shroff, H.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
[CrossRef]

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, PloS One 6, e20645 (2011).
[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, Science 313, 1642 (2006).
[CrossRef]

Spudich, J. A.

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, Nat. Methods 7, 377 (2010).
[CrossRef]

Stallinga, S.

B. Rieger and S. Stallinga, Chem. Phys. Chem. 15, 664 (2014).
[CrossRef]

S. Stallinga and B. Rieger, Opt. Express 18, 24461 (2010).
[CrossRef]

Subach, F. V.

M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
[CrossRef]

Testa, I.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

Verkhusha, V. V.

M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
[CrossRef]

von Middendorf, C.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

White, H.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, Proc. Natl. Acad. Sci. USA 104, 20308 (2007).
[CrossRef]

Wurm, C. A.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, Biophys. J. 99, 2686 (2010).
[CrossRef]

Yeung, E. S.

Y. Ma, M. R. Shortreed, and E. S. Yeung, Anal. Chem. 72, 4640 (2000).
[CrossRef]

Zhuang, X.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, Science 317, 1749 (2007).
[CrossRef]

M. J. Rust, M. Bates, and X. Zhuang, Nat. Methods 3, 793 (2006).
[CrossRef]

Anal. Chem. (1)

Y. Ma, M. R. Shortreed, and E. S. Yeung, Anal. Chem. 72, 4640 (2000).
[CrossRef]

Biometrika (1)

D. W. Scott, Biometrika 66, 605 (1979).
[CrossRef]

Biophys. J. (3)

M. S. Gunewardene, F. V. Subach, T. J. Gould, G. P. Penoncello, M. V. Gudheti, V. V. Verkhusha, and S. T. Hess, Biophys. J. 101, 1522 (2011).
[CrossRef]

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Figures (4)

Fig. 1.
Fig. 1.

Simulation results of the emitter position (a) and wavelength (b) precision as a function of signal photon count for three different background levels.

Fig. 2.
Fig. 2.

(a) Schematic overview of experimental setup. The sample is illuminated via a dichroic mirror (DM) and objective lens (OL), fluorescent light is captured and imaged onto the CCD via the tube lens (TL) and a 4f-relay of achromats L1 and L2 with the SLM in the pupil plane. (b) Example image of set of beads. (c) Average measured spot, after registration and upsampling, (d) average fitted spot, and (e) cross-sections of the average measured spot and average fitted spot.

Fig. 3.
Fig. 3.

Experimental results. (a) Precision of the x-position as a function of signal photon count showing agreement between the localization error determined from 25 consecutive fits with the mean precision according to the CRLB evaluated for the fitted parameter values. The inset shows a narrow distribution of background photon counts with an average of 5–6 photons per pixel. (b) Precision of the wavelength as a function of signal photon count showing agreement between the error determined from 25 consecutive fits with the mean precision according to the CRLB evaluated for the fitted parameter values. The inset shows the histogram over all fitted wavelength values for the individual fits, with a peak value around 590 nm, giving a small bias with the expected value of 612 nm.

Fig. 4.
Fig. 4.

(a) Image of a mixture of 8 different species of QDs (scalebar 10 μm). (b) Reference image of same area taken with a consumer color camera attached to the other side port of the microscope. Differences between (a) and (b) are due to differences in spectral sensitivity and blinking. (c) Histogram of found wavelengths from a time lapse measurements of a mixture of three species of QDs (QD605, QD655, QD705), with Gaussian fits of the three peaks [with widths of 600(12) nm, 635(10) nm, and 693(33) nm]. The magenta vertical lines at 623 nm and 648 nm indicate lines for classifying the three color species based on the estimated wavelength.

Equations (3)

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I(x,y)=jNj2πσ2e(xxj)2+(yyj)22σ2+ba2.
1(Δxem)2=j(xjxem)21(Δxj)2=jNjσ2(1I1(τj)),
1(Δλ)2=j[(xjλ)21(Δxj)2+(Njλ)21(ΔNj)2]=jNj[(jzσp)2(1I1(τj))+(1ηjηjλ)2(1I0(τj))].

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