Abstract

We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of solvent viscosity variations through the rotational correlation time of fluorescein in a multiwell plate and apply this technique to probe the microviscosity in live cells.

© 2004 Optical Society of America

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  1. A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, Biophys. J. 83, 1631 (2002).
    [CrossRef] [PubMed]
  2. J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
    [CrossRef]
  3. A. A. Newman, Glycerol (Morgan Grampian, London, 1968).
  4. J. R. Lakowicz, H. Cherek, B. P. Maliwal, and E. Gratton, Biochemistry 24, 376 (1985).
    [CrossRef] [PubMed]
  5. M. B. Mustafa, D. L. Tipton, M. D. Barkley, and P. S. Russo, Macromolecules 26, 370 (1993).
    [CrossRef]
  6. G. Porter, P. J. Sadkowski, and C. J. Tredwell, Chem. Phys. Lett. 49, 416 (1977).
  7. T. A. Smith, L. M. Bajada, and D. E. Dunstan, Macromolecules 35, 2736 (2002).
    [CrossRef]
  8. S. A. Rice and G. A. Kenney-Wallace, Chem. Phys. 47, 161 (1980).
  9. A. von Jena and H. E. Lessing, Chem. Phys. 40, 245 (1979).
  10. F. V. Bright, C. A. Monnig, and G. M. Hieftje, J. Appl. Phys. 61, 8 (1987).
    [CrossRef]
  11. A. S. Verkman, M. Armijo, and K. Fushimi, Biophys. Chem. 40, 117 (1991).
    [CrossRef] [PubMed]
  12. J. Jadzyn, G. Czechowski, and T. Stefaniak, J. Chem. Eng. Data 47, 978 (2002).
    [CrossRef]
  13. T. Lech, G. Czechowski, and J. Jadzyn, J. Chem. Eng. Data 46, 725 (2001).
    [CrossRef]
  14. K. Fushimi and A. S. Verkman, J. Cell Biol. 112, 719 (1991).
    [CrossRef] [PubMed]
  15. N. Periasamy, M. Armijo, and A. S. Verkman, Biochemistry 30, 11836 (1991).
    [CrossRef] [PubMed]
  16. J. A. Dix and A. S. Verkman, Biophys. J. 57, 231 (1990).
    [CrossRef] [PubMed]

2003

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

2002

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, Biophys. J. 83, 1631 (2002).
[CrossRef] [PubMed]

T. A. Smith, L. M. Bajada, and D. E. Dunstan, Macromolecules 35, 2736 (2002).
[CrossRef]

J. Jadzyn, G. Czechowski, and T. Stefaniak, J. Chem. Eng. Data 47, 978 (2002).
[CrossRef]

2001

T. Lech, G. Czechowski, and J. Jadzyn, J. Chem. Eng. Data 46, 725 (2001).
[CrossRef]

1993

M. B. Mustafa, D. L. Tipton, M. D. Barkley, and P. S. Russo, Macromolecules 26, 370 (1993).
[CrossRef]

1991

K. Fushimi and A. S. Verkman, J. Cell Biol. 112, 719 (1991).
[CrossRef] [PubMed]

N. Periasamy, M. Armijo, and A. S. Verkman, Biochemistry 30, 11836 (1991).
[CrossRef] [PubMed]

A. S. Verkman, M. Armijo, and K. Fushimi, Biophys. Chem. 40, 117 (1991).
[CrossRef] [PubMed]

1990

J. A. Dix and A. S. Verkman, Biophys. J. 57, 231 (1990).
[CrossRef] [PubMed]

1987

F. V. Bright, C. A. Monnig, and G. M. Hieftje, J. Appl. Phys. 61, 8 (1987).
[CrossRef]

1985

J. R. Lakowicz, H. Cherek, B. P. Maliwal, and E. Gratton, Biochemistry 24, 376 (1985).
[CrossRef] [PubMed]

1980

S. A. Rice and G. A. Kenney-Wallace, Chem. Phys. 47, 161 (1980).

1979

A. von Jena and H. E. Lessing, Chem. Phys. 40, 245 (1979).

1977

G. Porter, P. J. Sadkowski, and C. J. Tredwell, Chem. Phys. Lett. 49, 416 (1977).

1968

A. A. Newman, Glycerol (Morgan Grampian, London, 1968).

Armijo, M.

N. Periasamy, M. Armijo, and A. S. Verkman, Biochemistry 30, 11836 (1991).
[CrossRef] [PubMed]

A. S. Verkman, M. Armijo, and K. Fushimi, Biophys. Chem. 40, 117 (1991).
[CrossRef] [PubMed]

Arndt-Jovin, D. J.

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, Biophys. J. 83, 1631 (2002).
[CrossRef] [PubMed]

Bajada, L. M.

T. A. Smith, L. M. Bajada, and D. E. Dunstan, Macromolecules 35, 2736 (2002).
[CrossRef]

Barkley, M. D.

M. B. Mustafa, D. L. Tipton, M. D. Barkley, and P. S. Russo, Macromolecules 26, 370 (1993).
[CrossRef]

Bright, F. V.

F. V. Bright, C. A. Monnig, and G. M. Hieftje, J. Appl. Phys. 61, 8 (1987).
[CrossRef]

Cherek, H.

J. R. Lakowicz, H. Cherek, B. P. Maliwal, and E. Gratton, Biochemistry 24, 376 (1985).
[CrossRef] [PubMed]

Clayton, A. H. A.

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, Biophys. J. 83, 1631 (2002).
[CrossRef] [PubMed]

Czechowski, G.

J. Jadzyn, G. Czechowski, and T. Stefaniak, J. Chem. Eng. Data 47, 978 (2002).
[CrossRef]

T. Lech, G. Czechowski, and J. Jadzyn, J. Chem. Eng. Data 46, 725 (2001).
[CrossRef]

Davis, D. M.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Dix, J. A.

J. A. Dix and A. S. Verkman, Biophys. J. 57, 231 (1990).
[CrossRef] [PubMed]

Dunstan, D. E.

T. A. Smith, L. M. Bajada, and D. E. Dunstan, Macromolecules 35, 2736 (2002).
[CrossRef]

French, P. M. W.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Fushimi, K.

A. S. Verkman, M. Armijo, and K. Fushimi, Biophys. Chem. 40, 117 (1991).
[CrossRef] [PubMed]

K. Fushimi and A. S. Verkman, J. Cell Biol. 112, 719 (1991).
[CrossRef] [PubMed]

Gratton, E.

J. R. Lakowicz, H. Cherek, B. P. Maliwal, and E. Gratton, Biochemistry 24, 376 (1985).
[CrossRef] [PubMed]

Hanley, Q. S.

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, Biophys. J. 83, 1631 (2002).
[CrossRef] [PubMed]

Hieftje, G. M.

F. V. Bright, C. A. Monnig, and G. M. Hieftje, J. Appl. Phys. 61, 8 (1987).
[CrossRef]

Jadzyn, J.

J. Jadzyn, G. Czechowski, and T. Stefaniak, J. Chem. Eng. Data 47, 978 (2002).
[CrossRef]

T. Lech, G. Czechowski, and J. Jadzyn, J. Chem. Eng. Data 46, 725 (2001).
[CrossRef]

Jovin, T. M.

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, Biophys. J. 83, 1631 (2002).
[CrossRef] [PubMed]

Kenney-Wallace, G. A.

S. A. Rice and G. A. Kenney-Wallace, Chem. Phys. 47, 161 (1980).

Lakowicz, J. R.

J. R. Lakowicz, H. Cherek, B. P. Maliwal, and E. Gratton, Biochemistry 24, 376 (1985).
[CrossRef] [PubMed]

Lech, T.

T. Lech, G. Czechowski, and J. Jadzyn, J. Chem. Eng. Data 46, 725 (2001).
[CrossRef]

Lessing, H. E.

A. von Jena and H. E. Lessing, Chem. Phys. 40, 245 (1979).

Lévêque-Fort, S.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Maliwal, B. P.

J. R. Lakowicz, H. Cherek, B. P. Maliwal, and E. Gratton, Biochemistry 24, 376 (1985).
[CrossRef] [PubMed]

Monnig, C. A.

F. V. Bright, C. A. Monnig, and G. M. Hieftje, J. Appl. Phys. 61, 8 (1987).
[CrossRef]

Mustafa, M. B.

M. B. Mustafa, D. L. Tipton, M. D. Barkley, and P. S. Russo, Macromolecules 26, 370 (1993).
[CrossRef]

Newman, A. A.

A. A. Newman, Glycerol (Morgan Grampian, London, 1968).

Periasamy, N.

N. Periasamy, M. Armijo, and A. S. Verkman, Biochemistry 30, 11836 (1991).
[CrossRef] [PubMed]

Phillips, D.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Porter, G.

G. Porter, P. J. Sadkowski, and C. J. Tredwell, Chem. Phys. Lett. 49, 416 (1977).

Rice, S. A.

S. A. Rice and G. A. Kenney-Wallace, Chem. Phys. 47, 161 (1980).

Russo, P. S.

M. B. Mustafa, D. L. Tipton, M. D. Barkley, and P. S. Russo, Macromolecules 26, 370 (1993).
[CrossRef]

Sabharwal, Y.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Sadkowski, P. J.

G. Porter, P. J. Sadkowski, and C. J. Tredwell, Chem. Phys. Lett. 49, 416 (1977).

Siegel, J.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Smith, T. A.

T. A. Smith, L. M. Bajada, and D. E. Dunstan, Macromolecules 35, 2736 (2002).
[CrossRef]

Stefaniak, T.

J. Jadzyn, G. Czechowski, and T. Stefaniak, J. Chem. Eng. Data 47, 978 (2002).
[CrossRef]

Subramaniam, V.

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, Biophys. J. 83, 1631 (2002).
[CrossRef] [PubMed]

Suhling, K.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Tipton, D. L.

M. B. Mustafa, D. L. Tipton, M. D. Barkley, and P. S. Russo, Macromolecules 26, 370 (1993).
[CrossRef]

Tredwell, C. J.

G. Porter, P. J. Sadkowski, and C. J. Tredwell, Chem. Phys. Lett. 49, 416 (1977).

Verkman, A. S.

N. Periasamy, M. Armijo, and A. S. Verkman, Biochemistry 30, 11836 (1991).
[CrossRef] [PubMed]

A. S. Verkman, M. Armijo, and K. Fushimi, Biophys. Chem. 40, 117 (1991).
[CrossRef] [PubMed]

K. Fushimi and A. S. Verkman, J. Cell Biol. 112, 719 (1991).
[CrossRef] [PubMed]

J. A. Dix and A. S. Verkman, Biophys. J. 57, 231 (1990).
[CrossRef] [PubMed]

von Jena, A.

A. von Jena and H. E. Lessing, Chem. Phys. 40, 245 (1979).

Webb, S. E. D.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Biochemistry

J. R. Lakowicz, H. Cherek, B. P. Maliwal, and E. Gratton, Biochemistry 24, 376 (1985).
[CrossRef] [PubMed]

N. Periasamy, M. Armijo, and A. S. Verkman, Biochemistry 30, 11836 (1991).
[CrossRef] [PubMed]

Biophys. Chem.

A. S. Verkman, M. Armijo, and K. Fushimi, Biophys. Chem. 40, 117 (1991).
[CrossRef] [PubMed]

Biophys. J.

J. A. Dix and A. S. Verkman, Biophys. J. 57, 231 (1990).
[CrossRef] [PubMed]

A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin, Biophys. J. 83, 1631 (2002).
[CrossRef] [PubMed]

Chem. Phys.

S. A. Rice and G. A. Kenney-Wallace, Chem. Phys. 47, 161 (1980).

A. von Jena and H. E. Lessing, Chem. Phys. 40, 245 (1979).

Chem. Phys. Lett.

G. Porter, P. J. Sadkowski, and C. J. Tredwell, Chem. Phys. Lett. 49, 416 (1977).

J. Appl. Phys.

F. V. Bright, C. A. Monnig, and G. M. Hieftje, J. Appl. Phys. 61, 8 (1987).
[CrossRef]

J. Cell Biol.

K. Fushimi and A. S. Verkman, J. Cell Biol. 112, 719 (1991).
[CrossRef] [PubMed]

J. Chem. Eng. Data

J. Jadzyn, G. Czechowski, and T. Stefaniak, J. Chem. Eng. Data 47, 978 (2002).
[CrossRef]

J. Chem. Eng. Data

T. Lech, G. Czechowski, and J. Jadzyn, J. Chem. Eng. Data 46, 725 (2001).
[CrossRef]

Macromolecules

T. A. Smith, L. M. Bajada, and D. E. Dunstan, Macromolecules 35, 2736 (2002).
[CrossRef]

M. B. Mustafa, D. L. Tipton, M. D. Barkley, and P. S. Russo, Macromolecules 26, 370 (1993).
[CrossRef]

Rev. Sci. Instrum.

J. Siegel, K. Suhling, S. Lévêque-Fort, S. E. D. Webb, D. M. Davis, D. Phillips, Y. Sabharwal, and P. M. W. French, Rev. Sci. Instrum. 74, 182 (2003).
[CrossRef]

Other

A. A. Newman, Glycerol (Morgan Grampian, London, 1968).

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Figures (3)

Fig. 1
Fig. 1

Schematic of the polarization-resolved imager used to simultaneously acquire orthogonal polarization component images.2

Fig. 2
Fig. 2

(a) Schematic of mixtures of buffer and glycerol (in percent by weight) used to vary the viscosity of the fluorescein environment. (b) Fluorescence lifetime image with the polarization contribution removed. (c) Corresponding rotational correlation time image showing viscosity contrast over more than 1 order of magnitude. (d) Four representative time-resolved anisotropy decays averaged over a well. (e) Plot of the rotational correlation time θ versus the viscosity η, including our own time-correlated single-photon counting (TCSPC) data and data points from previous work.1,4,8-11 A straight line fit yields a fluorescein radius of 0.54 nm. (f) Plot of the background-corrected G factor of four representative wells as a function of time.

Fig. 3
Fig. 3

(a) FLIM map and (b) rotational correlation time map of CFSE-stained live B cell showing approximately homogeneous distributions throughout the cell. (c) Plot of anisotropy decay (with background subtraction) averaged over the entire cell.

Equations (3)

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

rt=It-B-GIt-BIt-B+2GIt-B,
rt=r0-rexp-tθ+r,
G=Ivv-BIhv-BIvh-BIhh-B1/2,

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