Abstract

Fluorescence correlation spectroscopy (FCS) is adapted for a new procedure to detect trace amounts of Escherichia coli in water. The present concept is based on convective diffusion rather than Brownian diffusion and employs confocal microscopy as in traditional FCS. With this system it is possible to detect concentrations as small as 1.5 × 105 E. coli per milliliter (2.5 × 10-16 M). This concentration corresponds to an ∼1.0-nM level of Rhodamine 6G dyes. A detailed analysis of the optical system is presented, and further improvements for the procedure are discussed.

© 2003 Optical Society of America

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References

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  1. G. J. Tortora, B. R. Funke, C. L. Case, in Microbiology, an Introduction, 5th ed. (Benjamin/Cummings, Redwood City, Calif.), p. 801.
  2. D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–709 (1972).
    [CrossRef]
  3. S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
    [CrossRef] [PubMed]
  4. H. Qian, E. L. Elson, “Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy,” Appl. Opt. 30, 1185–1195 (1991).
    [CrossRef] [PubMed]
  5. K. M. Berland, P. T. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
    [CrossRef] [PubMed]
  6. R. Rigler, “Fluorescence correlation, single molecule detection and large number screening applications in biotechnology,” J. Biotechnol. 41, 177–186 (1995).
    [CrossRef] [PubMed]
  7. N. L. Thompson, “Fluorescence correlation spectroscopy,” in Techniques, Vol. 1 of Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1971), pp. 337–378.
  8. J. Enderlein, “Theoretical study of detection of a dipole emitter through an objective with high numerical aperture,” Opt. Lett. 25, 634–636 (2000).
    [CrossRef]
  9. S. T. Hess, W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
    [CrossRef] [PubMed]
  10. D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and lamuinar flow,” Biopolymers 17, 361–376 (1978).
    [CrossRef]
  11. P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
    [CrossRef]
  12. B. H. Kunst, A. Schots, A. J. W. G. Visser, “Detection of flowing fluorescence particles in a microcapillary using fluorescence correlation spectroscopy,” Anal. Chem. 74, 5350–5357 (2002).
    [CrossRef] [PubMed]
  13. D. C. Lamb, A. Schenk, C. Rocker, C. Scalfi-Happ, G. U. Nienhaus, “Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection,” Biophys. J. 79, 1129–1138 (2000).
    [CrossRef] [PubMed]

2002

S. T. Hess, W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
[CrossRef] [PubMed]

B. H. Kunst, A. Schots, A. J. W. G. Visser, “Detection of flowing fluorescence particles in a microcapillary using fluorescence correlation spectroscopy,” Anal. Chem. 74, 5350–5357 (2002).
[CrossRef] [PubMed]

2000

D. C. Lamb, A. Schenk, C. Rocker, C. Scalfi-Happ, G. U. Nienhaus, “Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection,” Biophys. J. 79, 1129–1138 (2000).
[CrossRef] [PubMed]

J. Enderlein, “Theoretical study of detection of a dipole emitter through an objective with high numerical aperture,” Opt. Lett. 25, 634–636 (2000).
[CrossRef]

1999

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[CrossRef]

1997

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[CrossRef] [PubMed]

1995

K. M. Berland, P. T. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

R. Rigler, “Fluorescence correlation, single molecule detection and large number screening applications in biotechnology,” J. Biotechnol. 41, 177–186 (1995).
[CrossRef] [PubMed]

1991

1978

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and lamuinar flow,” Biopolymers 17, 361–376 (1978).
[CrossRef]

1972

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–709 (1972).
[CrossRef]

Berland, K. M.

K. M. Berland, P. T. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

Case, C. L.

G. J. Tortora, B. R. Funke, C. L. Case, in Microbiology, an Introduction, 5th ed. (Benjamin/Cummings, Redwood City, Calif.), p. 801.

Elson, E.

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–709 (1972).
[CrossRef]

Elson, E. L.

H. Qian, E. L. Elson, “Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy,” Appl. Opt. 30, 1185–1195 (1991).
[CrossRef] [PubMed]

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and lamuinar flow,” Biopolymers 17, 361–376 (1978).
[CrossRef]

Enderlein, J.

Funke, B. R.

G. J. Tortora, B. R. Funke, C. L. Case, in Microbiology, an Introduction, 5th ed. (Benjamin/Cummings, Redwood City, Calif.), p. 801.

Gratton, E.

K. M. Berland, P. T. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

Haupts, U.

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
[CrossRef] [PubMed]

Korlach, J.

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[CrossRef]

Kunst, B. H.

B. H. Kunst, A. Schots, A. J. W. G. Visser, “Detection of flowing fluorescence particles in a microcapillary using fluorescence correlation spectroscopy,” Anal. Chem. 74, 5350–5357 (2002).
[CrossRef] [PubMed]

Lamb, D. C.

D. C. Lamb, A. Schenk, C. Rocker, C. Scalfi-Happ, G. U. Nienhaus, “Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection,” Biophys. J. 79, 1129–1138 (2000).
[CrossRef] [PubMed]

Magde, D.

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and lamuinar flow,” Biopolymers 17, 361–376 (1978).
[CrossRef]

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–709 (1972).
[CrossRef]

Maiti, S.

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[CrossRef] [PubMed]

Nienhaus, G. U.

D. C. Lamb, A. Schenk, C. Rocker, C. Scalfi-Happ, G. U. Nienhaus, “Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection,” Biophys. J. 79, 1129–1138 (2000).
[CrossRef] [PubMed]

Qian, H.

Rigler, R.

R. Rigler, “Fluorescence correlation, single molecule detection and large number screening applications in biotechnology,” J. Biotechnol. 41, 177–186 (1995).
[CrossRef] [PubMed]

Rocker, C.

D. C. Lamb, A. Schenk, C. Rocker, C. Scalfi-Happ, G. U. Nienhaus, “Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection,” Biophys. J. 79, 1129–1138 (2000).
[CrossRef] [PubMed]

Scalfi-Happ, C.

D. C. Lamb, A. Schenk, C. Rocker, C. Scalfi-Happ, G. U. Nienhaus, “Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection,” Biophys. J. 79, 1129–1138 (2000).
[CrossRef] [PubMed]

Schenk, A.

D. C. Lamb, A. Schenk, C. Rocker, C. Scalfi-Happ, G. U. Nienhaus, “Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection,” Biophys. J. 79, 1129–1138 (2000).
[CrossRef] [PubMed]

Schots, A.

B. H. Kunst, A. Schots, A. J. W. G. Visser, “Detection of flowing fluorescence particles in a microcapillary using fluorescence correlation spectroscopy,” Anal. Chem. 74, 5350–5357 (2002).
[CrossRef] [PubMed]

Schwille, P.

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[CrossRef]

So, P. T.

K. M. Berland, P. T. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

Thompson, N. L.

N. L. Thompson, “Fluorescence correlation spectroscopy,” in Techniques, Vol. 1 of Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1971), pp. 337–378.

Tortora, G. J.

G. J. Tortora, B. R. Funke, C. L. Case, in Microbiology, an Introduction, 5th ed. (Benjamin/Cummings, Redwood City, Calif.), p. 801.

Visser, A. J. W. G.

B. H. Kunst, A. Schots, A. J. W. G. Visser, “Detection of flowing fluorescence particles in a microcapillary using fluorescence correlation spectroscopy,” Anal. Chem. 74, 5350–5357 (2002).
[CrossRef] [PubMed]

Webb, W. W.

S. T. Hess, W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
[CrossRef] [PubMed]

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[CrossRef]

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[CrossRef] [PubMed]

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and lamuinar flow,” Biopolymers 17, 361–376 (1978).
[CrossRef]

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–709 (1972).
[CrossRef]

Anal. Chem.

B. H. Kunst, A. Schots, A. J. W. G. Visser, “Detection of flowing fluorescence particles in a microcapillary using fluorescence correlation spectroscopy,” Anal. Chem. 74, 5350–5357 (2002).
[CrossRef] [PubMed]

Appl. Opt.

Biophys. J.

K. M. Berland, P. T. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995).
[CrossRef] [PubMed]

D. C. Lamb, A. Schenk, C. Rocker, C. Scalfi-Happ, G. U. Nienhaus, “Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection,” Biophys. J. 79, 1129–1138 (2000).
[CrossRef] [PubMed]

S. T. Hess, W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys. J. 83, 2300–2317 (2002).
[CrossRef] [PubMed]

Biopolymers

D. Magde, W. W. Webb, E. L. Elson, “Fluorescence correlation spectroscopy. III. Uniform translation and lamuinar flow,” Biopolymers 17, 361–376 (1978).
[CrossRef]

Cytometry

P. Schwille, J. Korlach, W. W. Webb, “Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes,” Cytometry 36, 176–182 (1999).
[CrossRef]

J. Biotechnol.

R. Rigler, “Fluorescence correlation, single molecule detection and large number screening applications in biotechnology,” J. Biotechnol. 41, 177–186 (1995).
[CrossRef] [PubMed]

Opt. Lett.

Phys. Rev. Lett.

D. Magde, E. Elson, W. W. Webb, “Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29, 705–709 (1972).
[CrossRef]

Proc. Natl. Acad. Sci. USA

S. Maiti, U. Haupts, W. W. Webb, “Fluorescence correlation spectroscopy: diagnostics for sparse molecules,” Proc. Natl. Acad. Sci. USA 94, 11753–11757 (1997).
[CrossRef] [PubMed]

Other

G. J. Tortora, B. R. Funke, C. L. Case, in Microbiology, an Introduction, 5th ed. (Benjamin/Cummings, Redwood City, Calif.), p. 801.

N. L. Thompson, “Fluorescence correlation spectroscopy,” in Techniques, Vol. 1 of Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum, New York, 1971), pp. 337–378.

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Figures (7)

Fig. 1
Fig. 1

Detailed schematic of the optical system composed of (1) argon-ion laser, (2) neutral-density filter, (3) bandpass filter, (4) diverging lens, (5) dichromatic mirror, (6) microscope objective lens, (7) stage, (8) filter, (9) converging lens, (10) pin hole, (11) optical fiber, (12) photon-counting unit, (13) digital correlator, (14) processor, (15) capillary tube or flow system, (16) reservoir, (17) pump.

Fig. 2
Fig. 2

Image of the focused laser beam. The beam waist is ∼20 μm.

Fig. 3
Fig. 3

Final incident energy F 6 as a function of various d 8 and d 10 values.

Fig. 4
Fig. 4

Autocorrelation functions of the sample with E. coli concentration of 1.5 × 107 ml (results from nine separate experiments).

Fig. 5
Fig. 5

Temporal fluctuation of fluorescence photon counts for various concentration of E. coli suspensions and water.

Fig. 6
Fig. 6

(a) Autocorrelation functions and (b) temporal fluctuation of fluorescence photon counts. Triangles, E. coli (1.5 × 105 ml-1) and Rhodamine 6G (1 nM); filled squares, Rhodamine 6G (1 nM); open diamonds, E. coli (1.5 × 105 ml-1).

Fig. 7
Fig. 7

Measured and corrected (theoretical) G(0) values as a function of E. coli concentrations.

Tables (1)

Tables Icon

Table 1 Examples of Parameter Values

Equations (13)

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

GτFtFτ+t-Ft2Ft2,
G0F2t-Ft2Ft2=σ2FF¯2,
G0=γ/N,
H1=τ1J0.
H2=τ2τ1J0.
H6=τ6τ5ρτ3τ2τ1J0,
F1=κH6,
F6=τ9τ8τ7τ4τ5515τ5488τ6515τ6488τ3τ2τ1ρd5+d6d7d8d9d102 κAJ0,
A=πCA22Ac1Ac2Ac3Ac4,
F6=TD κAJ0,
T=τ9τ8τ7τ4τ5τ62τ3τ2τ1ρ, D=d6d7d8d9d102.
Gτ=G0exp-τVω2,
G0=G0Meas×F¯TotalF¯Total-F¯Backgr2.

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