Abstract

Confocal optical microscopes offer unparalleled high sensitivity and three-dimensional (3D) imaging capability but require slow point-by-point scanning; they are inefficient for imaging moving objects. We propose a more efficient solution. Instead of indiscriminate scanning, we let the focus of the microscope pursue the object of interest such that no time is wasted on uninformative background, allowing us to visualize 3D trajectories of fluorescent nanoparticles in solution with millisecond temporal and 200nm spatial resolution.

© 2007 Optical Society of America

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References

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  1. M. Minsky, 'Microscopy apparatus,' U.S. patent 3,013,467 (November 7, 1957).
  2. G. Seisenberger, M. U. Ried, T. Endress, H. Buning, M. Hallek, and C. Brauchle, Science 294, 1929 (2001).
    [CrossRef] [PubMed]
  3. C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, Science 308, 1469 (2005).
    [CrossRef] [PubMed]
  4. X. Nan, P. A. Sims, P. Chen, and S. X. Xie, J. Phys. Chem. B 109, 24220 (2005).
    [CrossRef] [PubMed]
  5. J. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed. (Springer, 1995).
  6. J. E. Jureller, H. Y. Kim, and N. F. Scherer, Opt. Express 14, 3406 (2006).
    [CrossRef] [PubMed]
  7. H. Berg, Rev. Sci. Instrum. 42, 868 (1971).
    [CrossRef] [PubMed]
  8. A. E. Cohen and W. E. Moerner, Appl. Phys. Lett. 86, 093109 (2005).
    [CrossRef]
  9. H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, Appl. Phys. Lett. 22, 223901 (2006).
    [CrossRef]
  10. V. Levi, Q. Ruan, and E. Gratton, Biophys. J. 882919 (2005).
    [CrossRef] [PubMed]
  11. C. S. Xu, H. Cang, D. Montiel, and H. Yang, J. Phys. Chem. C 111, 32 (2007).
    [CrossRef]
  12. D. Montiel, H. Cang, and H. Yang, J. Phys. Chem. B 110, 19763 (2006).
    [CrossRef] [PubMed]
  13. M. Bruchez, M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos, Science 281, 2013 (1998).
    [CrossRef] [PubMed]
  14. W. C. W. Chan and S. M. Nie, Science 281, 2016 (1998).
    [CrossRef] [PubMed]

2007

C. S. Xu, H. Cang, D. Montiel, and H. Yang, J. Phys. Chem. C 111, 32 (2007).
[CrossRef]

2006

D. Montiel, H. Cang, and H. Yang, J. Phys. Chem. B 110, 19763 (2006).
[CrossRef] [PubMed]

J. E. Jureller, H. Y. Kim, and N. F. Scherer, Opt. Express 14, 3406 (2006).
[CrossRef] [PubMed]

H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, Appl. Phys. Lett. 22, 223901 (2006).
[CrossRef]

2005

V. Levi, Q. Ruan, and E. Gratton, Biophys. J. 882919 (2005).
[CrossRef] [PubMed]

A. E. Cohen and W. E. Moerner, Appl. Phys. Lett. 86, 093109 (2005).
[CrossRef]

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, Science 308, 1469 (2005).
[CrossRef] [PubMed]

X. Nan, P. A. Sims, P. Chen, and S. X. Xie, J. Phys. Chem. B 109, 24220 (2005).
[CrossRef] [PubMed]

2001

G. Seisenberger, M. U. Ried, T. Endress, H. Buning, M. Hallek, and C. Brauchle, Science 294, 1929 (2001).
[CrossRef] [PubMed]

1998

M. Bruchez, M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos, Science 281, 2013 (1998).
[CrossRef] [PubMed]

W. C. W. Chan and S. M. Nie, Science 281, 2016 (1998).
[CrossRef] [PubMed]

1971

H. Berg, Rev. Sci. Instrum. 42, 868 (1971).
[CrossRef] [PubMed]

Appl. Phys. Lett.

A. E. Cohen and W. E. Moerner, Appl. Phys. Lett. 86, 093109 (2005).
[CrossRef]

H. Cang, C. M. Wong, C. S. Xu, A. H. Rizvi, and H. Yang, Appl. Phys. Lett. 22, 223901 (2006).
[CrossRef]

Biophys. J.

V. Levi, Q. Ruan, and E. Gratton, Biophys. J. 882919 (2005).
[CrossRef] [PubMed]

J. Phys. Chem. B

X. Nan, P. A. Sims, P. Chen, and S. X. Xie, J. Phys. Chem. B 109, 24220 (2005).
[CrossRef] [PubMed]

D. Montiel, H. Cang, and H. Yang, J. Phys. Chem. B 110, 19763 (2006).
[CrossRef] [PubMed]

J. Phys. Chem. C

C. S. Xu, H. Cang, D. Montiel, and H. Yang, J. Phys. Chem. C 111, 32 (2007).
[CrossRef]

Opt. Express

Rev. Sci. Instrum.

H. Berg, Rev. Sci. Instrum. 42, 868 (1971).
[CrossRef] [PubMed]

Science

M. Bruchez, M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos, Science 281, 2013 (1998).
[CrossRef] [PubMed]

W. C. W. Chan and S. M. Nie, Science 281, 2016 (1998).
[CrossRef] [PubMed]

G. Seisenberger, M. U. Ried, T. Endress, H. Buning, M. Hallek, and C. Brauchle, Science 294, 1929 (2001).
[CrossRef] [PubMed]

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, Science 308, 1469 (2005).
[CrossRef] [PubMed]

Other

J. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed. (Springer, 1995).

M. Minsky, 'Microscopy apparatus,' U.S. patent 3,013,467 (November 7, 1957).

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Figures (3)

Fig. 1
Fig. 1

3D displacement detection. a, Axial, z direction, movement detection. b, Lateral, in-plane, movement detection. c, Schematic diagram of the optical layout. Abbreviation defined in text.

Fig. 2
Fig. 2

Immobilized nanosphere. a, Lateral scanning of an immobilized nanosphere. The dashed–dotted curve is the sum signal from the two x detectors, I 1 + I 2 . The dashed curve is the fit to a Gaussian. The solid curve is the normalized ratio between the two detectors, δ x = I 1 I 2 I 1 + I 2 . The dashed line is a guide for the eyes. b, Axial scanning of an immobilized nanosphere, normalized to 1 at maximum. The dashed curve is the sum signal from the four x y detectors, I x y = I 1 + I 2 + I 3 + I 4 , peaking at a difference position from that of the z detector, I z (dashed–dotted curve). The solid curve is the ratio of the two, I z I x y .

Fig. 3
Fig. 3

Diffusing nanoparticles. a, 3D trajectory of a 24 nm fluorescent nanosphere in a 70 % (wt) glycerol/water solution. The data were recorded at a 10 kHz sampling rate, but the time step in the figure is 1 ms for clarity. The total photon rate of the sphere as a function of time is shown in the bottom panel. b, 3D trajectory of a quantum dot in 87.2% (wt) glycerol/water solution. The total photon rate of the qdot is shown in the bottom panel. Notice the blinking in the trajectory marked by an arrow.

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