Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy

doi:10.1364/OE.14.005013

Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy

Markus Burkhardt and Petra Schwille »View Author Affiliations

Optics Express, Vol. 14, Issue 12, pp. 5013-5020 (2006)
http://dx.doi.org/10.1364/OE.14.005013

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Abstract

Fluorescence correlation spectroscopy (FCS) is carried out with an electron multiplying CCD (EMCCD). This new strategy is compared to standard detection by an avalanche photo diode showing good agreement with respect to the resulting autocorrelation curves. Applying different readout modes, a time resolution of 20 µs can be achieved, which is sufficient to resolve the diffusion of free dye in solution. The advantages of implementing EMCCD cameras in wide-field ultra low light imaging, as well as in multi-spot confocal laser scanning microscopy, can consequently also be exploited for spatially resolved FCS. First proof-of-principle FCS measurements with two excitation volumes demonstrate the advantage of the flexible CCD area detection.

OCIS Codes
(040.1520) Detectors : CCD, charge-coupled device
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(300.2530) Spectroscopy : Fluorescence, laser-induced

ToC Category:
Detectors

History
Original Manuscript: April 24, 2006
Revised Manuscript: May 19, 2006
Manuscript Accepted: May 19, 2006
Published: June 12, 2006

Virtual Issues
Vol. 1, Iss. 7 Virtual Journal for Biomedical Optics

Citation
Markus Burkhardt and Petra Schwille, "Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy," Opt. Express 14, 5013-5020 (2006)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-12-5013

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References

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972). [CrossRef]
R. Rigler, Ü. Mets, J. Widengren, and P. Kask, "Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion," Eur. Biophys. J. 22, 169-175 (1993). [CrossRef]
J. Widengren, R. Rigler, and Ü. Mets, "Triplet-state monitoring by fluorescence correlation spectroscopy," J. Fluoresc. 4, 255-258 (1994). [CrossRef]
U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, "Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy," Proc. Natl. Acad. Sci. U.S.A. 95, 13573-13578 (1998). [CrossRef]
P. Schwille, F. J. Meyer-Almes, and R. Rigler, "Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution," Biophys. J. 72, 1878-1886 (1997). [CrossRef] [PubMed]
M. Brinkmeier, K. Dorre, J. Stephan, and M. Eigen, "Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures," Anal. Chem. 71, 609-616 (1999). [CrossRef]
H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. Hard, and R. Rigler, "Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2x2 fan-out element," Appl. Opt. 41, 3336-3342 (2002). [CrossRef]
M. Gosch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, "Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array," J. Biomed. Opt. 9, 913-921 (2004). [CrossRef]

Anal. Chem.

M. Brinkmeier, K. Dorre, J. Stephan, and M. Eigen, "Two beam cross correlation: A method to characterize transport phenomena in micrometer-sized structures," Anal. Chem. 71, 609-616 (1999). [CrossRef]

Appl. Opt.

H. Blom, M. Johansson, A.-S. Hedman, L. Lundberg, A. Hanning, S. Hard, and R. Rigler, "Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2x2 fan-out element," Appl. Opt. 41, 3336-3342 (2002). [CrossRef]

Biophys. J.

P. Schwille, F. J. Meyer-Almes, and R. Rigler, "Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution," Biophys. J. 72, 1878-1886 (1997). [CrossRef] [PubMed]

Eur. Biophys. J.

R. Rigler, Ü. Mets, J. Widengren, and P. Kask, "Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion," Eur. Biophys. J. 22, 169-175 (1993). [CrossRef]

J. Biomed. Opt.

M. Gosch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, "Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array," J. Biomed. Opt. 9, 913-921 (2004). [CrossRef]

J. Fluoresc.

J. Widengren, R. Rigler, and Ü. Mets, "Triplet-state monitoring by fluorescence correlation spectroscopy," J. Fluoresc. 4, 255-258 (1994). [CrossRef]

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, 705-708 (1972). [CrossRef]

Proc. Natl. Acad. Sci. U.S.A.

U. Haupts, S. Maiti, P. Schwille, and W. W. Webb, "Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy," Proc. Natl. Acad. Sci. U.S.A. 95, 13573-13578 (1998). [CrossRef]

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