High countrate real-time FCS using F2Cor |
Optics Express, Vol. 21, Issue 20, pp. 23543-23555 (2013)
http://dx.doi.org/10.1364/OE.21.023543
Acrobat PDF (1408 KB)
Abstract
We present a fluorescence correlation spectroscopy setup based on a software correlator. The setup can process autocorrelation curves in real-time at countrate as high as 8MHz, with time resolution of 1µs. It uses the F2Cor autocorrelation algorithm, a low cost counting board and a desktop computer. Symmetrical normalization, which improves the signal to noise ratio of the FCS curve for large values of the lag-time, is adapted to the F2Cor algorithm. A new acquisition mode, which we call oscilloscope-mode, is presented. It takes advantage of the flexibility F2Cor, and proves to be very useful for optical setup adjustment. As an application of this setup, we performed FCS measurements on a reference tetramethylrhodamine solution at high concentration, up to 2.5µM, which extend to the micromolar range the concentration applicable in FCS, using a conventional optical setup. At such high countrates the FCS curves need to be corrected for dead-time of the photo-detector, which was done successfully.
© 2013 Optical Society of America
1. Introduction
1. E. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974). [CrossRef]
5. T. A. Laurence, S. Fore, and T. Huser, “Fast, flexible algorithm for calculating photon correlations,” Opt. Lett. 31(6), 829–831 (2006). [CrossRef] [PubMed]
9. L. L. Yang, H. Y. Lee, M. K. Wang, X. Y. Lin, K. H. Hsu, Y. R. Chang, W. Fann, and J. D. White, “Real-time data acquisition incorporating high-speed software correlator for single-molecule spectroscopy,” J. Microsc. 234(3), 302–310 (2009). [CrossRef] [PubMed]
10. K. Schätzel, “Correlation techniques in dynamic light scattering,” Appl. Phys. B 42, 193–213 (1987). [CrossRef]
12. J. S. Eid, J. D. Muller, and E. Gratton, “Data acquisition card for fluctuation correlation spectroscopy allowing full access to the detected photon sequence,” Rev. Sci. Instrum. 71(2), 361–368 (2000). [CrossRef]
6. D. Magatti and F. Ferri, “Fast multi-tau real-time software correlator for dynamic light scattering,” Appl. Opt. 40(24), 4011–4021 (2001). [CrossRef] [PubMed]
7. D. Magatti and F. Ferri, “25 ns software correlator for photon and fluorescence correlation spectroscopy,” Rev. Sci. Instrum. 74(2), 1135–1144 (2003). [CrossRef]
5. T. A. Laurence, S. Fore, and T. Huser, “Fast, flexible algorithm for calculating photon correlations,” Opt. Lett. 31(6), 829–831 (2006). [CrossRef] [PubMed]
13. E. Schaub, “F2Cor: fast 2-stage correlation algorithm for FCS and DLS,” Opt. Express 20(3), 2184–2195 (2012). [CrossRef] [PubMed]
13. E. Schaub, “F2Cor: fast 2-stage correlation algorithm for FCS and DLS,” Opt. Express 20(3), 2184–2195 (2012). [CrossRef] [PubMed]
2. Setup
12. J. S. Eid, J. D. Muller, and E. Gratton, “Data acquisition card for fluctuation correlation spectroscopy allowing full access to the detected photon sequence,” Rev. Sci. Instrum. 71(2), 361–368 (2000). [CrossRef]
14. DMA Performance Improvements for TIO-based Devices,” http://digital.ni.com/public.nsf/allkb/1B64310FAE9007C086256A1D006D9BBF.
15. S. A. Kim, K. G. Heinze, and P. Schwille, “Fluorescence correlation spectroscopy in living cells,” Nat. Methods 4(11), 963–973 (2007). [CrossRef] [PubMed]
13. E. Schaub, “F2Cor: fast 2-stage correlation algorithm for FCS and DLS,” Opt. Express 20(3), 2184–2195 (2012). [CrossRef] [PubMed]
3. Symmetrical normalization
16. K. Schätzel, M. Drewel, and S. Stimac, “Photon correlation measurements at large lag times: improving statistical accuracy,” J. Mod. Opt. 35(4), 711–718 (1988). [CrossRef]
13. E. Schaub, “F2Cor: fast 2-stage correlation algorithm for FCS and DLS,” Opt. Express 20(3), 2184–2195 (2012). [CrossRef] [PubMed]
13. E. Schaub, “F2Cor: fast 2-stage correlation algorithm for FCS and DLS,” Opt. Express 20(3), 2184–2195 (2012). [CrossRef] [PubMed]
16. K. Schätzel, M. Drewel, and S. Stimac, “Photon correlation measurements at large lag times: improving statistical accuracy,” J. Mod. Opt. 35(4), 711–718 (1988). [CrossRef]
17. T. Wohland, R. Rigler, and H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80(6), 2987–2999 (2001). [CrossRef] [PubMed]
4. Oscilloscope mode FCS
5. Measurements on a reference dye
18. D. E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974). [CrossRef]
19. L. Neri, S. Tudisco, F. Musumeci, A. Scordino, G. Fallica, M. Mazzillo, and M. Zimbone, “Note: Dead time causes and correction method for single photon avalanche diode devices,” Rev. Sci. Instrum. 81(8), 086102–086103 (2010). [CrossRef] [PubMed]
6. Discussion and prospects
20. J. Wenger, D. Gérard, P. F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, “Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations,” Opt. Express 14(25), 12206–12216 (2006). [CrossRef] [PubMed]
15. S. A. Kim, K. G. Heinze, and P. Schwille, “Fluorescence correlation spectroscopy in living cells,” Nat. Methods 4(11), 963–973 (2007). [CrossRef] [PubMed]
7. Conclusion
Acknowledgments
References and links
1. | E. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974). [CrossRef] |
2. | D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974). [CrossRef] [PubMed] |
3. | P. Schwille and J. Ries, “Principles and applications of fluorescence correlation spectroscopy (FCS),” in Biophotonics: Spectroscopy, Imaging, Sensing, and Manipulation (Springer, 2011), pp. 63–85. |
4. | ALV-5000 multiple tau digital correlator reference manual (ALV gmbh, 1993). |
5. | T. A. Laurence, S. Fore, and T. Huser, “Fast, flexible algorithm for calculating photon correlations,” Opt. Lett. 31(6), 829–831 (2006). [CrossRef] [PubMed] |
6. | D. Magatti and F. Ferri, “Fast multi-tau real-time software correlator for dynamic light scattering,” Appl. Opt. 40(24), 4011–4021 (2001). [CrossRef] [PubMed] |
7. | D. Magatti and F. Ferri, “25 ns software correlator for photon and fluorescence correlation spectroscopy,” Rev. Sci. Instrum. 74(2), 1135–1144 (2003). [CrossRef] |
8. | M. Wahl, I. Gregor, M. Patting, and J. Enderlein, “Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting,” Opt. Express 11(26), 3583–3591 (2003). [CrossRef] [PubMed] |
9. | L. L. Yang, H. Y. Lee, M. K. Wang, X. Y. Lin, K. H. Hsu, Y. R. Chang, W. Fann, and J. D. White, “Real-time data acquisition incorporating high-speed software correlator for single-molecule spectroscopy,” J. Microsc. 234(3), 302–310 (2009). [CrossRef] [PubMed] |
10. | K. Schätzel, “Correlation techniques in dynamic light scattering,” Appl. Phys. B 42, 193–213 (1987). [CrossRef] |
11. | W. Liu, J. Shen, and X. Sun, “Design of multiple-tau photon correlation system implemented by FPGA ,” Software and Systems, 410-414 (2008). |
12. | J. S. Eid, J. D. Muller, and E. Gratton, “Data acquisition card for fluctuation correlation spectroscopy allowing full access to the detected photon sequence,” Rev. Sci. Instrum. 71(2), 361–368 (2000). [CrossRef] |
13. | E. Schaub, “F2Cor: fast 2-stage correlation algorithm for FCS and DLS,” Opt. Express 20(3), 2184–2195 (2012). [CrossRef] [PubMed] |
14. | DMA Performance Improvements for TIO-based Devices,” http://digital.ni.com/public.nsf/allkb/1B64310FAE9007C086256A1D006D9BBF. |
15. | S. A. Kim, K. G. Heinze, and P. Schwille, “Fluorescence correlation spectroscopy in living cells,” Nat. Methods 4(11), 963–973 (2007). [CrossRef] [PubMed] |
16. | K. Schätzel, M. Drewel, and S. Stimac, “Photon correlation measurements at large lag times: improving statistical accuracy,” J. Mod. Opt. 35(4), 711–718 (1988). [CrossRef] |
17. | T. Wohland, R. Rigler, and H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80(6), 2987–2999 (2001). [CrossRef] [PubMed] |
18. | D. E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974). [CrossRef] |
19. | L. Neri, S. Tudisco, F. Musumeci, A. Scordino, G. Fallica, M. Mazzillo, and M. Zimbone, “Note: Dead time causes and correction method for single photon avalanche diode devices,” Rev. Sci. Instrum. 81(8), 086102–086103 (2010). [CrossRef] [PubMed] |
20. | J. Wenger, D. Gérard, P. F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, “Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations,” Opt. Express 14(25), 12206–12216 (2006). [CrossRef] [PubMed] |
21. | Z. Petrasek and P. Schwille, “Scanning fluorescence correlation spectroscopy,” in Single Molecules and Nanotechnology (Springer, 2008), pp. 83–105. |
OCIS Codes
(070.4550) Fourier optics and signal processing : Correlators
(120.6200) Instrumentation, measurement, and metrology : Spectrometers and spectroscopic instrumentation
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(070.7145) Fourier optics and signal processing : Ultrafast processing
ToC Category:
Spectroscopy
History
Original Manuscript: July 16, 2013
Revised Manuscript: September 13, 2013
Manuscript Accepted: September 19, 2013
Published: September 26, 2013
Citation
Emmanuel Schaub, "High countrate real-time FCS using F2Cor," Opt. Express 21, 23543-23555 (2013)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-21-20-23543
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References
- E. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers13(1), 1–27 (1974). [CrossRef]
- D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers13(1), 29–61 (1974). [CrossRef] [PubMed]
- P. Schwille and J. Ries, “Principles and applications of fluorescence correlation spectroscopy (FCS),” in Biophotonics: Spectroscopy, Imaging, Sensing, and Manipulation (Springer, 2011), pp. 63–85.
- ALV-5000 multiple tau digital correlator reference manual (ALV gmbh, 1993).
- T. A. Laurence, S. Fore, and T. Huser, “Fast, flexible algorithm for calculating photon correlations,” Opt. Lett.31(6), 829–831 (2006). [CrossRef] [PubMed]
- D. Magatti and F. Ferri, “Fast multi-tau real-time software correlator for dynamic light scattering,” Appl. Opt.40(24), 4011–4021 (2001). [CrossRef] [PubMed]
- D. Magatti and F. Ferri, “25 ns software correlator for photon and fluorescence correlation spectroscopy,” Rev. Sci. Instrum.74(2), 1135–1144 (2003). [CrossRef]
- M. Wahl, I. Gregor, M. Patting, and J. Enderlein, “Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting,” Opt. Express11(26), 3583–3591 (2003). [CrossRef] [PubMed]
- L. L. Yang, H. Y. Lee, M. K. Wang, X. Y. Lin, K. H. Hsu, Y. R. Chang, W. Fann, and J. D. White, “Real-time data acquisition incorporating high-speed software correlator for single-molecule spectroscopy,” J. Microsc.234(3), 302–310 (2009). [CrossRef] [PubMed]
- K. Schätzel, “Correlation techniques in dynamic light scattering,” Appl. Phys. B42, 193–213 (1987). [CrossRef]
- W. Liu, J. Shen, and X. Sun, “Design of multiple-tau photon correlation system implemented by FPGA,” Software and Systems, 410-414 (2008).
- J. S. Eid, J. D. Muller, and E. Gratton, “Data acquisition card for fluctuation correlation spectroscopy allowing full access to the detected photon sequence,” Rev. Sci. Instrum.71(2), 361–368 (2000). [CrossRef]
- E. Schaub, “F2Cor: fast 2-stage correlation algorithm for FCS and DLS,” Opt. Express20(3), 2184–2195 (2012). [CrossRef] [PubMed]
- DMA Performance Improvements for TIO-based Devices,” http://digital.ni.com/public.nsf/allkb/1B64310FAE9007C086256A1D006D9BBF .
- S. A. Kim, K. G. Heinze, and P. Schwille, “Fluorescence correlation spectroscopy in living cells,” Nat. Methods4(11), 963–973 (2007). [CrossRef] [PubMed]
- K. Schätzel, M. Drewel, and S. Stimac, “Photon correlation measurements at large lag times: improving statistical accuracy,” J. Mod. Opt.35(4), 711–718 (1988). [CrossRef]
- T. Wohland, R. Rigler, and H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J.80(6), 2987–2999 (2001). [CrossRef] [PubMed]
- D. E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A10(6), 1938–1945 (1974). [CrossRef]
- L. Neri, S. Tudisco, F. Musumeci, A. Scordino, G. Fallica, M. Mazzillo, and M. Zimbone, “Note: Dead time causes and correction method for single photon avalanche diode devices,” Rev. Sci. Instrum.81(8), 086102–086103 (2010). [CrossRef] [PubMed]
- J. Wenger, D. Gérard, P. F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, “Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations,” Opt. Express14(25), 12206–12216 (2006). [CrossRef] [PubMed]
- Z. Petrasek and P. Schwille, “Scanning fluorescence correlation spectroscopy,” in Single Molecules and Nanotechnology (Springer, 2008), pp. 83–105.
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