## EMCCD-based spectrally resolved fluorescence correlation spectroscopy |

Optics Express, Vol. 18, Issue 23, pp. 23818-23828 (2010)

http://dx.doi.org/10.1364/OE.18.023818

Acrobat PDF (1103 KB)

### Abstract

We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.

© 2010 OSA

## 1. Introduction

1. M. Ehrenberg and R. Rigler, “Rotational Brownian Motion and fluorescence intensity fluctuation,” Chem. Phys. **4**(3), 390–401 (1974). [CrossRef]

4. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers **13**(1), 29–61 (1974). [CrossRef] [PubMed]

5. K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods **29**(1), 74–85 (2003). [CrossRef] [PubMed]

9. J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. **99**(36), 13368–13379 (1995). [CrossRef]

10. J. Rika and T. Binkert, “Direct measurement of a distinct correlation function by fluorescence cross correlation,” Phys. Rev. A **39**(5), 2646–2652 (1989). [CrossRef] [PubMed]

11. P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. **72**(4), 1878–1886 (1997). [CrossRef] [PubMed]

12. M. Burkhardt, K. G. Heinze, and P. Schwille, “Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform,” Opt. Lett. **30**(17), 2266–2268 (2005). [CrossRef] [PubMed]

15. M. J. R. Previte, S. Pelet, K. H. Kim, C. Buehler, and P. T. C. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. **80**(9), 3277–3284 (2008). [CrossRef] [PubMed]

16. M. Burkhardt and P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express **14**(12), 5013–5020 (2006). [CrossRef] [PubMed]

17. B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. **78**(10), 3444–3451 (2006). [CrossRef] [PubMed]

18. D. Boening, T. W. Groemer, and J. Klingauf, “Applicability of an EM-CCD for spatially resolved TIR-ICS,” Opt. Express **18**(13), 13516–13528 (2010). [CrossRef] [PubMed]

22. T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express **18**(10), 10627–10641 (2010). [CrossRef] [PubMed]

*in vitro*and

*in vivo*, to study photophysical properties of fluorophores with good spectral and temporal resolution and to measure molecular interactions in living cells with spectrally optimized settings.

## 2. Experimental setup

### 2.1 Hardware

*In vivo*experiments were carried out at 37°C using an incubation chamber enclosing the microscope stage and body (EMBL workshops).

### 2.2 Software

*k*=

*l*) and cross-correlation analysis (

*k*≠

*l*) according to Eq. (1):

23. K. Schätzel, “Noise on photon correlation data. I. Autocorrelation functions,” Quantum Opt. **2**(4), 287–305 (1990). [CrossRef]

24. M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. **298**(4), 677–689 (2000). [CrossRef] [PubMed]

*N*is the average number of molecules in the focus,

*f*

_{1}the amplitude fraction of the first component,

_{i}the diffusion correlation time and the anomaly parameter, respectively, of component

*i*with its diffusion coefficient

*D*and its radius of gyration

_{i}*R*, and

_{i}*e*

^{2}radius

*z*

_{0}and

*w*

_{0}of the focal volume.

*T*(or an average number

*N·T*) is in such a dark state with a typical lifetime of τ

_{blink}, the amplitude of the correlation function is increased accordingly. The normalized cross-correlation amplitude is defined as the ratio of the cross- and the geometrically averaged autocorrelation amplitudes as obtained from the fits of Eq. (2) to the data and is computed as

## 3. Sample preparation

26. K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. **324**(2), 849–854 (2004). [CrossRef] [PubMed]

*in vivo*experiments, cells were seeded in 8 well Nunc LabTek chambered coverglasses (Thermo Fischer Scientific, Langenselbold, Germany) with phenol red-free RPMI medium. For

*in vitro*experiments, Alexa 488 and quantum dots (QDs) with emission maxima at 525, 565, 605 and 655 nm referred to as QD 525, 565, 605, and 655, respectively (Invitrogen Germany, Karlsruhe, Germany), were dissolved in water and dispensed in 8 well LabTek chambered coverglasses, too, at the concentrations as given below.

## 4. Characterization of the setup

### 4.1 Camera characterization

27. J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. **95**(11), 5385–5398 (2008). [CrossRef] [PubMed]

^{–}) in this pixel. Their number is converted by the camera electronics into pixel gray values given in analog-to-digital units (ADU). In order to transform pixel values in a camera image back to counted photo-electrons the corresponding conversion factor

*f*

_{conv}must be determined. The number of photo-electrons results from a counting process and thus obeys Poisson statistics so that its average equals its variance. Thus, for homogeneous illumination the standard deviation of the pixel values σ

_{ADU}as averaged over the whole image depends on the average pixel value μ

_{ADU}, the conversion factor and the readout noise σ

_{RO}according to [28] as shown in Eq. (4):We recorded the detector signal at homogeneous low light illumination for a range of integration times at a CMG of 30. The slope of the pixel value variance as plotted against the average pixel value for all integration times yielded a conversion factor of 0.052 e

^{–}/ADU.

^{1/2}gave an RMS readout noise of 0.6 e

^{–}, i.e., small enough for single photon counting and FCS.

19. G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. **38**(6), 813–828 (2009). [CrossRef] [PubMed]

### 4.2 Spectral calibration

29. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science **307**(5709), 538–544 (2005). [CrossRef] [PubMed]

### 4.3 Comparison of FCS measurements acquired with the EMCCD and with APDs

*f*

_{1}= 1) of Eq. (2) to the data very similar diffusion correlation times could be obtained, showing that we get substantially the same results with both setups.

*N*resulting from the fits of Eq. (2) to the data showed an almost linear dependence on the concentration, see Fig. 3C, supported by a fit of a power law to a double-logarithmic representation of the data that yielded a slope of 1.06±0.04. Only for relatively small concentrations below 2 nM we observed a deviation from linearity due to an increasing influence of the background signal.

## 5. Spectrally resolved FCS measurements

### 5.1 FCS of a mixture of QDs

^{2}≈5%. Therefore, fits with a single freely diffusive component and an exponential blinking contribution, i.e. α = 1 and

*f*

_{1}= 1 in Eq. (2), were justified and resulted in diffusion correlation times that increased with increasing maximum emission wavelength, see Fig. 4B. For an estimation of the expected spectral dependence of the diffusion correlation times we used the radii of gyration of 6, 7, 8 and 9 nm (values provided by the manufacturer) for QD 525, 565, 605 and 655, respectively, including core, shell and polymer coating of the QDs. We estimated the additional chromatic contribution based on the fact that a constant pinhole size was used for the complete spectrum and on the assumption that the focal radius

*w*

_{0}depends on the illumination and the detection wavelengths λ

_{ill/det}according to[25

25. T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. **3**(1), 49–61 (2002). [CrossRef]

30. S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. **77**(7), 2235–2242 (2005). [CrossRef] [PubMed]

### 5.2 Spectrally resolved fluorescence intermittence of QD 655

_{diff}and

*N*showed a spectral dependence stronger than the prediction and also τ

_{blink}and θ

_{blink}varied with the wavelength. Therefore, we repeated the fitting with the number of molecules and diffusion correlation times following the estimated spectral dependence (solid lines in Fig. 5A and B) whereas the blinking correlation time and the fraction of non-fluorescent particles were free parameters, see Fig. 5C, D and E. Both parameters decreased with increasing wavelength and approached a plateau above 655 nm. That way we made use of the spectral resolution to obtain information that is not accessible with a conventional FCS setup.

30. S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. **77**(7), 2235–2242 (2005). [CrossRef] [PubMed]

33. J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. **102**(40), 14284–14289 (2005). [CrossRef] [PubMed]

### 5.3 Spectrally resolved FCCS in living cells

26. K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. **324**(2), 849–854 (2004). [CrossRef] [PubMed]

## 6. Conclusion

34. A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. **101**(24), 8936–8941 (2004). [CrossRef] [PubMed]

35. S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. **11**(2), 024012–024012 (2006). [CrossRef] [PubMed]

18. D. Boening, T. W. Groemer, and J. Klingauf, “Applicability of an EM-CCD for spatially resolved TIR-ICS,” Opt. Express **18**(13), 13516–13528 (2010). [CrossRef] [PubMed]

22. T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express **18**(10), 10627–10641 (2010). [CrossRef] [PubMed]

## Acknowledgements

## References and links

1. | M. Ehrenberg and R. Rigler, “Rotational Brownian Motion and fluorescence intensity fluctuation,” Chem. Phys. |

2. | E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers |

3. | D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlations spectroscopy,” Phys. Rev. Lett. |

4. | D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers |

5. | K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods |

6. | M. Gösch and R. Rigler, “Fluorescence correlation spectroscopy of molecular motions and kinetics,” Adv. Drug Deliv. Rev. |

7. | J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. |

8. | M. Wachsmuth, and K. Weisshart, “Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells,” in |

9. | J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. |

10. | J. Rika and T. Binkert, “Direct measurement of a distinct correlation function by fluorescence cross correlation,” Phys. Rev. A |

11. | P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. |

12. | M. Burkhardt, K. G. Heinze, and P. Schwille, “Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform,” Opt. Lett. |

13. | K. G. Heinze, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: one step further in following higher order molecular complex formation,” Biophys. J. |

14. | L. C. Hwang, M. Leutenegger, M. Gösch, T. Lasser, P. Rigler, W. Meier, and T. Wohland, “Prism-based multicolor fluorescence correlation spectrometer,” Opt. Lett. |

15. | M. J. R. Previte, S. Pelet, K. H. Kim, C. Buehler, and P. T. C. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. |

16. | M. Burkhardt and P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express |

17. | B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. |

18. | D. Boening, T. W. Groemer, and J. Klingauf, “Applicability of an EM-CCD for spatially resolved TIR-ICS,” Opt. Express |

19. | G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. |

20. | D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. |

21. | D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. |

22. | T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express |

23. | K. Schätzel, “Noise on photon correlation data. I. Autocorrelation functions,” Quantum Opt. |

24. | M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. |

25. | T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. |

26. | K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. |

27. | J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. |

28. | F. Christen, K. Kuijken, D. Baade, C. Cavadore, S. Deiries, and O. Iwert, “Fast Conversion Factor (Gain) Measurement of a CCD Using Images With Vertical Gradient,” in |

29. | X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science |

30. | S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. |

31. | P. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. |

32. | R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: Correlation functions versus distributions of on- and off-times,” J. Chem. Phys. |

33. | J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. |

34. | A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. |

35. | S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. |

36. | B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially Resolved Total Internal Reflection Fluorescence Correlation Microscopy Using an Electron Multiplying Charge-Coupled Device Camera,” Analytical Chemistry (2007). |

**OCIS Codes**

(040.1520) Detectors : CCD, charge-coupled device

(180.1790) Microscopy : Confocal microscopy

(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence

**ToC Category:**

Spectroscopy

**History**

Original Manuscript: July 13, 2010

Revised Manuscript: September 29, 2010

Manuscript Accepted: October 1, 2010

Published: October 27, 2010

**Virtual Issues**

Vol. 6, Iss. 1 *Virtual Journal for Biomedical Optics*

**Citation**

Felix Bestvater, Zahir Seghiri, Moon Sik Kang, Nadine Gröner, Ji Young Lee, Kang-Bin Im, and Malte Wachsmuth, "EMCCD-based spectrally resolved fluorescence correlation spectroscopy," Opt. Express **18**, 23818-23828 (2010)

http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-18-23-23818

Sort: Year | Journal | Reset

### References

- M. Ehrenberg and R. Rigler, “Rotational Brownian Motion and fluorescence intensity fluctuation,” Chem. Phys. 4(3), 390–401 (1974). [CrossRef]
- E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974). [CrossRef]
- D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlations spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972). [CrossRef]
- D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974). [CrossRef] [PubMed]
- K. Bacia and P. Schwille, “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy,” Methods 29(1), 74–85 (2003). [CrossRef] [PubMed]
- M. Gösch and R. Rigler, “Fluorescence correlation spectroscopy of molecular motions and kinetics,” Adv. Drug Deliv. Rev. 57(1), 169–190 (2005). [CrossRef]
- J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008). [CrossRef]
- M. Wachsmuth, and K. Weisshart, “Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells,” in Imaging Cellular and Molecular Biological Functions (Springer Verlag, Heidelberg, 2007).
- J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995). [CrossRef]
- J. Rika and T. Binkert, “Direct measurement of a distinct correlation function by fluorescence cross correlation,” Phys. Rev. A 39(5), 2646–2652 (1989). [CrossRef] [PubMed]
- P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997). [CrossRef] [PubMed]
- M. Burkhardt, K. G. Heinze, and P. Schwille, “Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform,” Opt. Lett. 30(17), 2266–2268 (2005). [CrossRef] [PubMed]
- K. G. Heinze, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: one step further in following higher order molecular complex formation,” Biophys. J. 86(1), 506–516 (2004). [CrossRef]
- L. C. Hwang, M. Leutenegger, M. Gösch, T. Lasser, P. Rigler, W. Meier, and T. Wohland, “Prism-based multicolor fluorescence correlation spectrometer,” Opt. Lett. 31(9), 1310–1312 (2006). [CrossRef] [PubMed]
- M. J. R. Previte, S. Pelet, K. H. Kim, C. Buehler, and P. T. C. So, “Spectrally resolved fluorescence correlation spectroscopy based on global analysis,” Anal. Chem. 80(9), 3277–3284 (2008). [CrossRef] [PubMed]
- M. Burkhardt and P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006). [CrossRef] [PubMed]
- B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006). [CrossRef] [PubMed]
- D. Boening, T. W. Groemer, and J. Klingauf, “Applicability of an EM-CCD for spatially resolved TIR-ICS,” Opt. Express 18(13), 13516–13528 (2010). [CrossRef] [PubMed]
- G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009). [CrossRef] [PubMed]
- D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009). [CrossRef] [PubMed]
- D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006). [CrossRef] [PubMed]
- T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express 18(10), 10627–10641 (2010). [CrossRef] [PubMed]
- K. Schätzel, “Noise on photon correlation data. I. Autocorrelation functions,” Quantum Opt. 2(4), 287–305 (1990). [CrossRef]
- M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000). [CrossRef] [PubMed]
- T. Weidemann, M. Wachsmuth, M. Tewes, K. Rippe, and J. Langowski, “Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy,” Single Mol. 3(1), 49–61 (2002). [CrossRef]
- K. Saito, I. Wada, M. Tamura, and M. Kinjo, “Direct detection of caspase-3 activation in single live cells by cross-correlation analysis,” Biochem. Biophys. Res. Commun. 324(2), 849–854 (2004). [CrossRef] [PubMed]
- J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008). [CrossRef] [PubMed]
- F. Christen, K. Kuijken, D. Baade, C. Cavadore, S. Deiries, and O. Iwert, “Fast Conversion Factor (Gain) Measurement of a CCD Using Images With Vertical Gradient,” in Scientific detectors for astronomy 2005 (Springer Netherlands, 2006).
- X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307(5709), 538–544 (2005). [CrossRef] [PubMed]
- S. Doose, J. M. Tsay, F. Pinaud, and S. Weiss, “Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy,” Anal. Chem. 77(7), 2235–2242 (2005). [CrossRef] [PubMed]
- P. Frantsuzov, M. Kuno, B. Janko, and R. A. Marcus, “Universal emission intermittency in quantum dots, nanorods and nanowires,” Nat. Phys. 4(5), 519–522 (2008). [CrossRef]
- R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: Correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003). [CrossRef]
- J. Yao, D. R. Larson, H. D. Vishwasrao, W. R. Zipfel, and W. W. Webb, “Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution,” Proc. Natl. Acad. Sci. U.S.A. 102(40), 14284–14289 (2005). [CrossRef] [PubMed]
- A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proc. Natl. Acad. Sci. U.S.A. 101(24), 8936–8941 (2004). [CrossRef] [PubMed]
- S. Rüttinger, R. Macdonald, B. Krämer, F. Koberling, M. Roos, and E. Hildt, “Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy,” J. Biomed. Opt. 11(2), 024012–024012 (2006). [CrossRef] [PubMed]
- B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially Resolved Total Internal Reflection Fluorescence Correlation Microscopy Using an Electron Multiplying Charge-Coupled Device Camera,” Analytical Chemistry (2007).

## Cited By |
Alert me when this paper is cited |

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article | Next Article »

OSA is a member of CrossRef.