Abstract

We designed a fluorescence correlation spectroscopy (FCS) system for measurements on surfaces. The system consists of an objective-type total internal reflection fluorescence (TIRF) microscopy setup, adapted to measure FCS. Here, the fluorescence exciting evanescent wave is generated by epi-illumination through the periphery of a high NA oil-immersion objective. The main advantages with respect to conventional FCS systems are an improvement in terms of counts per molecule (cpm) and a high signal to background ratio. This is demonstrated by investigating diffusion as well as binding and release of single molecules on a glass surface. Furthermore, the size and shape of the molecule detection efficiency (MDE) function was calculated, using a wave-vectorial approach and taking into account the influence of the dielectric interface on the emission properties of fluorophores.

© 2005 Optical Society of America

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References

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    [CrossRef]

Anal. Chem.

R. L. Hansen and J. M. Harris, �??Total Internal Reflection Fluorescence Correlation Spectroscopy for Counting Molecules at Solid/Liquid Interfaces,�?? Anal. Chem. 70, 2565�??2575 (1998).
[CrossRef] [PubMed]

Biophys. J.

N. L. Thompson, T. P. Burghardt, and D. Axelrod, �??Measuring Surface Dynamics of Biomolecules by Total Internal- Reflection Fluorescence with Photobleaching Recovery or Correlation Spectroscopy,�?? Biophys. J. 33, 435�??454 (1981).
[CrossRef] [PubMed]

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, �??High count rates with total internal reflection fluorescence correlation spectroscopy,�?? Biophys. J. 88, L1�??L3 (2005).
[CrossRef]

A. M. Lieto, R. C. Cush, and N. L. Thompson, �??Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,�?? Biophys. J. 85, 3294�??3302 (2003).
[CrossRef] [PubMed]

Cell. Mol. Biol.

R. Brock and T. M. Jovin, �??Fluorescence correlation microscopy (FCM) - Fluorescence correlation spectroscopy (FCS) taken into the cell,�?? Cell. Mol. Biol. 44, 847�??856 (1998).
[PubMed]

Chem. Phys.

L. Edman, Z. Foldes-Papp, S.Wennmalm, and R. Rigler, �??The fluctuating enzyme: a single molecule approach,�?? Chem. Phys. 247, 11�??22 (1999).
[CrossRef]

Chem. Phys. Lett.

J. Enderlein, �??Fluorescence detection of single molecules near a solution/glass interface - an electrodynamic analysis,�?? Chem. Phys. Lett. 308, 263�??266 (1999).
[CrossRef]

Drug Discov. Today

M. Auer, K. J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, �??Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS,�?? Drug Discov. Today 3, 457�??465 (1998).

Eur. Biophys. J. Biophys. Lett.

R. Rigler, U. Mets, J. Widengren, and P. Kask, �??Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion,�?? Eur. Biophys. J. Biophys. Lett. 22, 169�??175 (1993).
[CrossRef]

J. Opt. Soc. Am.

J. Opt. Soc. Am. B

Phys. Rev. Lett.

D. Magde, W. W. Webb, and E. Elson, �??Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy,�?? Phys. Rev. Lett. 29, 705�??& (1972).
[CrossRef]

Proc. Natl. Acad. Sci. USA

U. Kettling, A. Koltermann, P. Schwille, and M. Eigen, �??Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy,�?? Proc. Natl. Acad. Sci. USA 95, 1416�??1420 (1998).
[CrossRef] [PubMed]

Proc. Roy. Soc. A

B. Richards and E. Wolf, �??Electromagnetic Diffraction in Optical Systems .2. Structure of the Image Field in an Aplanatic System,�?? Proc. Roy. Soc. A 253, 358�??379 (1959).
[CrossRef]

Rep. Progr. Phys.

O. Krichevsky and G. Bonnet, �??Fluorescence correlation spectroscopy: the technique and its applications,�?? Rep. Progr. Phys. 65, 251�??297 (2002).
[CrossRef]

Science

S. Weiss, �??Fluorescence Spectroscopy of Single Biomolecules,�?? Science 283, 1676�??1683 (1999).
[CrossRef] [PubMed]

Talanta

C. T. Culbertson, S. C. Jacobson, and J. M. Ramsey, �??Diffusion coefficient measurements in microfluidic devices,�?? Talanta 56, 365�??373 (2002).
[CrossRef]

Topics in Fluorescence Spectroscopy

N. L. Thompson, �??Fluorescence Correlation Spectroscopy,�?? in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed., vol. 1 (Plenum Press, New York, 1991).

Topics in Fluorescence Spectroscopy: Bio

D. Axelrod, E. H. Hellen, and R. M. Fulbright, �??Total Internal Reflection Fluorescence,�?? in Topics in Fluorescence Spectroscopy: Biochemical Applications, J. R. Lakowicz, ed., vol. 3 (Plenum Press, 1992).

Tr. Cell Biol.

D. Toomre and D. J. Manstein, �??Lighting up the cell surface with evanescent wave microscopy,�?? Tr. Cell Biol. 11, 298�??303 (2001).
[CrossRef]

Traffic

D. Axelrod, �??Total internal reflection fluorescence microscopy in cell biology,�?? Traffic 2, 764�??774 (2001).
[CrossRef] [PubMed]

Other

R. Rigler and E. L. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications (Springer, Berlin, 2001).
[CrossRef]

K. Hassler, M. Leutenegger, M. Gösch, T. Lasser, Laboratoire d�??Optique Biomédicale, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland are preparing a manuscript to be called �??Mathematical Models for Total Internal Reflection Fluorescence Correlation Spectroscopy�??.

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Figures (4)

Fig. 1.
Fig. 1.

Schematic representation of the ‘objective-type TIR-FCS’ setup (left). L1 - L4: lenses; F1, F2: fluorescence filters; GP: glass plate; D: dichroic mirror; Obj: microscope objective; bfp: back focal plane of the objective; MS: motorized scanning stage. An enlargement of the ray-path inside the microscope objective (right). d: evanescent wave depth; Θ c : critical angle.

Fig. 2.
Fig. 2.

The normalized MDEs for confocal FCS (left) and TIR-FCS (right). The confocal FCS case was calculated for a 1.15 NA, 40 × water-immersion objective. For the TIR-FCS case a 1.45 NA, 100 × oil-immersion objective was considered. A diameter of the pinhole (core of the fiber) of 50μm was assumed in both cases. The excitation wavelength was 488 nm and the fluorescence emission wavelength was 542 nm.

Fig. 3.
Fig. 3.

Typical time trace for single rhodamine 6G molecules binding to a microscope slide (left). The right picture shows an enlargement of the two highest bursts. The binning time is 100μs.

Fig. 4.
Fig. 4.

Autocorrelation for diffusing rhodamine 6G molecules (upper left) and time trace (right). The overall measurement time was 30 s. Fitting the data with the model represented by equation 6 yields the following parameters: N = 1.2, τ z = 21.1μs, ω =0.38, p=15.4%, τt =1.6μs and cpm = 1.77 MHz. The red curve represents the fit to the autocorrelation data.

Tables (1)

Tables Icon

Table 1. Parameter estimates for measurements with different pinhole diameters. pd: diameter of the pinhole. D: diffusion coefficient. ωt : theoretically obtained structure parameter. For other symbols refer to the discussion of equation 6. The parameter τ t was fixed to 1μs.

Equations (7)

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

MDE ( r ) = c CEF ( r ) I ( r ) .
d = λ 4 π ( n 2 2 sin 2 ( θ ) n 1 2 ) 1 2 .
CEF ( r ) = P ( z ) S circ ( q a ) PSF ( q q , z ) d q .
W n V MDE n ( r ) d r ,
MDE a ( x , y , z ) = exp ( 2 ( x 2 + y 2 ) ω x y 2 ) exp ( z h ) .
G ( τ ) = 1 + γ N [ 1 + p 1 p exp ( τ τ t ) ] ( 1 + τ ω 2 τ z ) 1
× [ ( 1 τ 2 τ z ) w ( i τ 4 τ z ) + τ π τ z ] .

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