Abstract

We show that the position of a fluorescent nanoparticle can be measured in three dimensions with subnanometer precision and 100-ms temporal resolution by use of standard epifluorescence video imaging in off-focus mode. The particle can be tracked without feedback in a volume of at least 40 µm×60 µm×3 µm. With the technique presented, the structure–mobility relationship of 216-nm latex particle in a porous polymer network was studied in three dimensions.

© 2003 Optical Society of America

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  1. M. De Brabander, G. Geuens, R. Nuydens, M. Moeremans, and J. De Mey, Cytobios 43, 273 (1985).
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
  7. M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
    [CrossRef]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
  14. C. Tischer, S. Altmann, S. Fisinger, E. H. K. Stelzer, J. K. H. Hörber, and E.-L. Florin, Appl. Phys. Lett. 79, 3878 (2001).
    [CrossRef]

2001

R. Yasuda, H. Noji, M. Yoshida, K. Kinosita, and H. Itoh, Nature 410, 898 (2001).
[CrossRef] [PubMed]

C. M. Ajo-Franklin, L. Kam, and S. G. Boxer, Proc. Natl. Acad. Sci. USA 98, 13643 (2001).
[CrossRef]

C. Tischer, S. Altmann, S. Fisinger, E. H. K. Stelzer, J. K. H. Hörber, and E.-L. Florin, Appl. Phys. Lett. 79, 3878 (2001).
[CrossRef]

2000

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

1997

M. J. Saxton and K. Jacobson, Annu. Rev. Biophys. Biomol. Struct. 26, 373 (1997).
[CrossRef]

1996

J. C. Crocker and D. G. Grier, J. Colloid. Interf. Sci. 179, 298 (1996).
[CrossRef]

1994

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

1993

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. (Oxford) 169, 391 (1993).
[CrossRef]

1989

1986

N. Bobroff, Rev. Sci. Instrum. 57, 1152 (1986).
[CrossRef]

1985

M. De Brabander, G. Geuens, R. Nuydens, M. Moeremans, and J. De Mey, Cytobios 43, 273 (1985).

D. H. Burns, J. B. Callis, G. D. Christian, and E. R. Davidson, Appl. Opt. 24, 154 (1985).
[CrossRef]

Agard, D. A.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy of Living Cells in Culture, Part B, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), pp. 353–377.
[CrossRef]

Ajo-Franklin, C. M.

C. M. Ajo-Franklin, L. Kam, and S. G. Boxer, Proc. Natl. Acad. Sci. USA 98, 13643 (2001).
[CrossRef]

Altmann, S.

C. Tischer, S. Altmann, S. Fisinger, E. H. K. Stelzer, J. K. H. Hörber, and E.-L. Florin, Appl. Phys. Lett. 79, 3878 (2001).
[CrossRef]

Bobroff, N.

N. Bobroff, Rev. Sci. Instrum. 57, 1152 (1986).
[CrossRef]

Boxer, S. G.

C. M. Ajo-Franklin, L. Kam, and S. G. Boxer, Proc. Natl. Acad. Sci. USA 98, 13643 (2001).
[CrossRef]

Burns, D. H.

Callis, J. B.

Christian, G. D.

Cremer, C.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. (Oxford) 169, 391 (1993).
[CrossRef]

Crocker, J. C.

J. C. Crocker and D. G. Grier, J. Colloid. Interf. Sci. 179, 298 (1996).
[CrossRef]

Davidson, E. R.

De Brabander, M.

M. De Brabander, G. Geuens, R. Nuydens, M. Moeremans, and J. De Mey, Cytobios 43, 273 (1985).

De Mey, J.

M. De Brabander, G. Geuens, R. Nuydens, M. Moeremans, and J. De Mey, Cytobios 43, 273 (1985).

Fisinger, S.

C. Tischer, S. Altmann, S. Fisinger, E. H. K. Stelzer, J. K. H. Hörber, and E.-L. Florin, Appl. Phys. Lett. 79, 3878 (2001).
[CrossRef]

Florin, E.-L.

C. Tischer, S. Altmann, S. Fisinger, E. H. K. Stelzer, J. K. H. Hörber, and E.-L. Florin, Appl. Phys. Lett. 79, 3878 (2001).
[CrossRef]

A. Pralle and E.-L. Florin, in Atomic Force Microscopy in Cell Biology, B. P. Jena and J. K. H. Hörber, eds., Vol. 68 of Methods in Cell Biology (Academic, San Diego, Calif., 2002), pp. 193–213.
[CrossRef]

Geuens, G.

M. De Brabander, G. Geuens, R. Nuydens, M. Moeremans, and J. De Mey, Cytobios 43, 273 (1985).

Gibson, S. F.

Grier, D. G.

J. C. Crocker and D. G. Grier, J. Colloid. Interf. Sci. 179, 298 (1996).
[CrossRef]

Hell, S.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. (Oxford) 169, 391 (1993).
[CrossRef]

Hell, S. W.

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Hiraoka, Y.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy of Living Cells in Culture, Part B, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), pp. 353–377.
[CrossRef]

Hörber, J. K. H.

C. Tischer, S. Altmann, S. Fisinger, E. H. K. Stelzer, J. K. H. Hörber, and E.-L. Florin, Appl. Phys. Lett. 79, 3878 (2001).
[CrossRef]

Itoh, H.

R. Yasuda, H. Noji, M. Yoshida, K. Kinosita, and H. Itoh, Nature 410, 898 (2001).
[CrossRef] [PubMed]

Jacobson, K.

M. J. Saxton and K. Jacobson, Annu. Rev. Biophys. Biomol. Struct. 26, 373 (1997).
[CrossRef]

Kam, L.

C. M. Ajo-Franklin, L. Kam, and S. G. Boxer, Proc. Natl. Acad. Sci. USA 98, 13643 (2001).
[CrossRef]

Kao, H. P.

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

Kinosita, K.

R. Yasuda, H. Noji, M. Yoshida, K. Kinosita, and H. Itoh, Nature 410, 898 (2001).
[CrossRef] [PubMed]

Lanni, F.

Moeremans, M.

M. De Brabander, G. Geuens, R. Nuydens, M. Moeremans, and J. De Mey, Cytobios 43, 273 (1985).

Nagorni, M.

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Noji, H.

R. Yasuda, H. Noji, M. Yoshida, K. Kinosita, and H. Itoh, Nature 410, 898 (2001).
[CrossRef] [PubMed]

Nuydens, R.

M. De Brabander, G. Geuens, R. Nuydens, M. Moeremans, and J. De Mey, Cytobios 43, 273 (1985).

Pralle, A.

A. Pralle and E.-L. Florin, in Atomic Force Microscopy in Cell Biology, B. P. Jena and J. K. H. Hörber, eds., Vol. 68 of Methods in Cell Biology (Academic, San Diego, Calif., 2002), pp. 193–213.
[CrossRef]

Reiner, G.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. (Oxford) 169, 391 (1993).
[CrossRef]

Saxton, M. J.

M. J. Saxton and K. Jacobson, Annu. Rev. Biophys. Biomol. Struct. 26, 373 (1997).
[CrossRef]

Schmidt, M.

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

Sedat, J. W.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy of Living Cells in Culture, Part B, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), pp. 353–377.
[CrossRef]

Shaw, P.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy of Living Cells in Culture, Part B, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), pp. 353–377.
[CrossRef]

Stelzer, E. H. K.

C. Tischer, S. Altmann, S. Fisinger, E. H. K. Stelzer, J. K. H. Hörber, and E.-L. Florin, Appl. Phys. Lett. 79, 3878 (2001).
[CrossRef]

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. (Oxford) 169, 391 (1993).
[CrossRef]

Tischer, C.

C. Tischer, S. Altmann, S. Fisinger, E. H. K. Stelzer, J. K. H. Hörber, and E.-L. Florin, Appl. Phys. Lett. 79, 3878 (2001).
[CrossRef]

Verkman, A. S.

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

Yasuda, R.

R. Yasuda, H. Noji, M. Yoshida, K. Kinosita, and H. Itoh, Nature 410, 898 (2001).
[CrossRef] [PubMed]

Yoshida, M.

R. Yasuda, H. Noji, M. Yoshida, K. Kinosita, and H. Itoh, Nature 410, 898 (2001).
[CrossRef] [PubMed]

Annu. Rev. Biophys. Biomol. Struct.

M. J. Saxton and K. Jacobson, Annu. Rev. Biophys. Biomol. Struct. 26, 373 (1997).
[CrossRef]

Appl. Opt.

Appl. Phys. Lett.

C. Tischer, S. Altmann, S. Fisinger, E. H. K. Stelzer, J. K. H. Hörber, and E.-L. Florin, Appl. Phys. Lett. 79, 3878 (2001).
[CrossRef]

Biophys. J.

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).
[CrossRef] [PubMed]

Cytobios

M. De Brabander, G. Geuens, R. Nuydens, M. Moeremans, and J. De Mey, Cytobios 43, 273 (1985).

J. Colloid. Interf. Sci.

J. C. Crocker and D. G. Grier, J. Colloid. Interf. Sci. 179, 298 (1996).
[CrossRef]

J. Microsc. (Oxford)

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, J. Microsc. (Oxford) 169, 391 (1993).
[CrossRef]

J. Opt. Soc. Am. A

Nature

R. Yasuda, H. Noji, M. Yoshida, K. Kinosita, and H. Itoh, Nature 410, 898 (2001).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. USA

C. M. Ajo-Franklin, L. Kam, and S. G. Boxer, Proc. Natl. Acad. Sci. USA 98, 13643 (2001).
[CrossRef]

Rev. Sci. Instrum.

M. Schmidt, M. Nagorni, and S. W. Hell, Rev. Sci. Instrum. 71, 2742 (2000).
[CrossRef]

N. Bobroff, Rev. Sci. Instrum. 57, 1152 (1986).
[CrossRef]

Other

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, in Fluorescence Microscopy of Living Cells in Culture, Part B, D. L. Taylor and Y. Wang, eds., Vol. 30 of Methods in Cell Biology (Academic, San Diego, Calif., 1989), pp. 353–377.
[CrossRef]

A. Pralle and E.-L. Florin, in Atomic Force Microscopy in Cell Biology, B. P. Jena and J. K. H. Hörber, eds., Vol. 68 of Methods in Cell Biology (Academic, San Diego, Calif., 2002), pp. 193–213.
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Images of a 216-nm fluorescent bead embedded in an agarose gel at different distances z from the focal plane (left). In all measurements, actual distances from the focal plane were corrected for refractive-index mismatch between the immersion oil (1.518) and the agarose (1.331).12 Within the z range 0.5 µm,3.5 µm, the radius r0 of the outermost ring in the images scaled linearly with z (right). The bead was fixed 2 µm above the coverslip surface.

Fig. 2
Fig. 2

Position tracking of a 216-nm bead embedded in agarose gel. The bead position was modulated with (a) a square wave of peak-to-peak amplitude 2 nm and period 5 s in the lateral direction and (b) amplitude 5 nm and period 10 s in the axial direction. The average position standard deviations are (a) 0.8 nm (integration time 62 ms) and (b) 0.9 nm (integration time 106 ms). The average z position was 1 µm for both cases.

Fig. 3
Fig. 3

Precision of the 3-D SPT as a function of the bead’s axial position. Single standard deviations σx,σy, and σz of the x,y, and z positions of a stationary 216-nm bead embedded in an agarose gel are shown. The camera integration time for image acquisition was 106 ms. The standard deviations were calculated from 20 data points.

Fig. 4
Fig. 4

Hindered diffusion of a 216-nm bead in an agar gel network. The dots represent accessible positions of the bead center; vacant volume elements of tubular shape contain agar filaments (see also inset). The bead position was determined in 2000 successive images containing two to three rings recorded with 7.5 frames/s and integration time of 100 ms. The total volume explored by the beam within the recording time of 267 s was 0.34 µm×0.57 µm×0.15 µm.

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