Abstract

Fluorescence Correlation Spectroscopy (FCS) in cells often suffers from artifacts caused by bright aggregates or vesicles, depletion of fluorophores or bleaching of a fluorescent background. The common practice of manually discarding distorted curves is time consuming and subjective. Here we demonstrate the feasibility of automated FCS data analysis with efficient rejection of corrupted parts of the signal. As test systems we use a solution of fluorescent molecules, contaminated with bright fluorescent beads, as well as cells expressing a fluorescent protein (ICA512-EGFP), which partitions into bright secretory granules. This approach improves the accuracy of FCS measurements in biological samples, extends its applicability to especially challenging systems and greatly simplifies and accelerates the data analysis.

© 2010 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  13. M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
    [CrossRef] [PubMed]
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    [CrossRef]

2009

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

A. Tcherniak, C. Reznik, S. Link, and C. F. Landes, "Fluorescence correlation spectroscopy: criteria for analysis in complex systems," Anal. Chem. 81(2), 746-754 (2009).
[CrossRef]

2008

J. Ries and P. Schwille, "New Concepts for Fluorescence Correlation Spectroscopy on Membranes," Phys. Chem. Chem. Phys. 10(24), 3487-3497 (2008).
[CrossRef] [PubMed]

C. C. Guet, L. Bruneaux, T. L. Min, D. Siegal-Gaskins, I. Figueroa, T. Emonet, and P. Cluzel, "Minimally invasive determination of mRNA concentration in single living bacteria," Nucleic Acids Res. 36(12), e73 (2008).
[CrossRef] [PubMed]

2007

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements," ChemPhysChem 8(3), 433-443 (2007).
[CrossRef] [PubMed]

S. Kim, K. Heinze, and P. Schwille, "Fluorescence correlation spectroscopy in living cells," Nat. Methods 4(11), 963-974 (2007).
[CrossRef] [PubMed]

2006

K. Bacia, S. Kim, and P. Schwille, "Fluorescence cross-correlation spectroscopy in living cells," Nat. Methods 3(2), 83-89 (2006).
[CrossRef] [PubMed]

G. Meacci, J. Ries, E. Fischer-Friedrich, N. Kahya, P. Schwille, and K. Kruse, "Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy," Phys. Biol. 3(4), 255-263 (2006).
[CrossRef]

2004

M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
[CrossRef] [PubMed]

2003

K. Bacia and P. Schwille, "A dynamic view of cellular processes by in vivo fluorescence auto-and crosscorrelation spectroscopy," Methods 29(1), 74-85 (2003).
[CrossRef] [PubMed]

2001

1992

M. Asfari, D. Janjic, P. Meda, G. Li, P. A. Halban, and C. B. Wollheim, "Establishment of 2-mercaptoethanoldependent differentiated insulin-secreting cell lines," Endocrinology 130(1), 167-178 (1992).
[CrossRef] [PubMed]

1974

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13 (1), 1-27 (1974).
[CrossRef]

Altkruger, A.

M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
[CrossRef] [PubMed]

Asfari, M.

M. Asfari, D. Janjic, P. Meda, G. Li, P. A. Halban, and C. B. Wollheim, "Establishment of 2-mercaptoethanoldependent differentiated insulin-secreting cell lines," Endocrinology 130(1), 167-178 (1992).
[CrossRef] [PubMed]

Bacia, K.

K. Bacia, S. Kim, and P. Schwille, "Fluorescence cross-correlation spectroscopy in living cells," Nat. Methods 3(2), 83-89 (2006).
[CrossRef] [PubMed]

K. Bacia and P. Schwille, "A dynamic view of cellular processes by in vivo fluorescence auto-and crosscorrelation spectroscopy," Methods 29(1), 74-85 (2003).
[CrossRef] [PubMed]

Brand, M.

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

Bruneaux, L.

C. C. Guet, L. Bruneaux, T. L. Min, D. Siegal-Gaskins, I. Figueroa, T. Emonet, and P. Cluzel, "Minimally invasive determination of mRNA concentration in single living bacteria," Nucleic Acids Res. 36(12), e73 (2008).
[CrossRef] [PubMed]

Burkhardt, M.

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

Cluzel, P.

C. C. Guet, L. Bruneaux, T. L. Min, D. Siegal-Gaskins, I. Figueroa, T. Emonet, and P. Cluzel, "Minimally invasive determination of mRNA concentration in single living bacteria," Nucleic Acids Res. 36(12), e73 (2008).
[CrossRef] [PubMed]

Dertinger, T.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements," ChemPhysChem 8(3), 433-443 (2007).
[CrossRef] [PubMed]

Elson, E. L.

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13 (1), 1-27 (1974).
[CrossRef]

Emonet, T.

C. C. Guet, L. Bruneaux, T. L. Min, D. Siegal-Gaskins, I. Figueroa, T. Emonet, and P. Cluzel, "Minimally invasive determination of mRNA concentration in single living bacteria," Nucleic Acids Res. 36(12), e73 (2008).
[CrossRef] [PubMed]

Enderlein, J.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements," ChemPhysChem 8(3), 433-443 (2007).
[CrossRef] [PubMed]

Ferri, F.

Figueroa, I.

C. C. Guet, L. Bruneaux, T. L. Min, D. Siegal-Gaskins, I. Figueroa, T. Emonet, and P. Cluzel, "Minimally invasive determination of mRNA concentration in single living bacteria," Nucleic Acids Res. 36(12), e73 (2008).
[CrossRef] [PubMed]

Fischer-Friedrich, E.

G. Meacci, J. Ries, E. Fischer-Friedrich, N. Kahya, P. Schwille, and K. Kruse, "Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy," Phys. Biol. 3(4), 255-263 (2006).
[CrossRef]

Gregor, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements," ChemPhysChem 8(3), 433-443 (2007).
[CrossRef] [PubMed]

Guet, C. C.

C. C. Guet, L. Bruneaux, T. L. Min, D. Siegal-Gaskins, I. Figueroa, T. Emonet, and P. Cluzel, "Minimally invasive determination of mRNA concentration in single living bacteria," Nucleic Acids Res. 36(12), e73 (2008).
[CrossRef] [PubMed]

Halban, P. A.

M. Asfari, D. Janjic, P. Meda, G. Li, P. A. Halban, and C. B. Wollheim, "Establishment of 2-mercaptoethanoldependent differentiated insulin-secreting cell lines," Endocrinology 130(1), 167-178 (1992).
[CrossRef] [PubMed]

Hartmann, R.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements," ChemPhysChem 8(3), 433-443 (2007).
[CrossRef] [PubMed]

Heinze, K.

S. Kim, K. Heinze, and P. Schwille, "Fluorescence correlation spectroscopy in living cells," Nat. Methods 4(11), 963-974 (2007).
[CrossRef] [PubMed]

Janjic, D.

M. Asfari, D. Janjic, P. Meda, G. Li, P. A. Halban, and C. B. Wollheim, "Establishment of 2-mercaptoethanoldependent differentiated insulin-secreting cell lines," Endocrinology 130(1), 167-178 (1992).
[CrossRef] [PubMed]

Kahya, N.

G. Meacci, J. Ries, E. Fischer-Friedrich, N. Kahya, P. Schwille, and K. Kruse, "Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy," Phys. Biol. 3(4), 255-263 (2006).
[CrossRef]

Kim, S.

S. Kim, K. Heinze, and P. Schwille, "Fluorescence correlation spectroscopy in living cells," Nat. Methods 4(11), 963-974 (2007).
[CrossRef] [PubMed]

K. Bacia, S. Kim, and P. Schwille, "Fluorescence cross-correlation spectroscopy in living cells," Nat. Methods 3(2), 83-89 (2006).
[CrossRef] [PubMed]

Knoch, K. P.

M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
[CrossRef] [PubMed]

Kruse, K.

G. Meacci, J. Ries, E. Fischer-Friedrich, N. Kahya, P. Schwille, and K. Kruse, "Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy," Phys. Biol. 3(4), 255-263 (2006).
[CrossRef]

Landes, C. F.

A. Tcherniak, C. Reznik, S. Link, and C. F. Landes, "Fluorescence correlation spectroscopy: criteria for analysis in complex systems," Anal. Chem. 81(2), 746-754 (2009).
[CrossRef]

Li, G.

M. Asfari, D. Janjic, P. Meda, G. Li, P. A. Halban, and C. B. Wollheim, "Establishment of 2-mercaptoethanoldependent differentiated insulin-secreting cell lines," Endocrinology 130(1), 167-178 (1992).
[CrossRef] [PubMed]

Link, S.

A. Tcherniak, C. Reznik, S. Link, and C. F. Landes, "Fluorescence correlation spectroscopy: criteria for analysis in complex systems," Anal. Chem. 81(2), 746-754 (2009).
[CrossRef]

Magatti, D.

Magde, D.

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13 (1), 1-27 (1974).
[CrossRef]

Meacci, G.

G. Meacci, J. Ries, E. Fischer-Friedrich, N. Kahya, P. Schwille, and K. Kruse, "Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy," Phys. Biol. 3(4), 255-263 (2006).
[CrossRef]

Meda, P.

M. Asfari, D. Janjic, P. Meda, G. Li, P. A. Halban, and C. B. Wollheim, "Establishment of 2-mercaptoethanoldependent differentiated insulin-secreting cell lines," Endocrinology 130(1), 167-178 (1992).
[CrossRef] [PubMed]

Min, T. L.

C. C. Guet, L. Bruneaux, T. L. Min, D. Siegal-Gaskins, I. Figueroa, T. Emonet, and P. Cluzel, "Minimally invasive determination of mRNA concentration in single living bacteria," Nucleic Acids Res. 36(12), e73 (2008).
[CrossRef] [PubMed]

Muller, S.

M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
[CrossRef] [PubMed]

Mziaut, H.

M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
[CrossRef] [PubMed]

Nowak, M.

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

Ouwendijk, J.

M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
[CrossRef] [PubMed]

Pacheco, V.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements," ChemPhysChem 8(3), 433-443 (2007).
[CrossRef] [PubMed]

Petr’asek, Z.

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

Reznik, C.

A. Tcherniak, C. Reznik, S. Link, and C. F. Landes, "Fluorescence correlation spectroscopy: criteria for analysis in complex systems," Anal. Chem. 81(2), 746-754 (2009).
[CrossRef]

Ries, J.

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

J. Ries and P. Schwille, "New Concepts for Fluorescence Correlation Spectroscopy on Membranes," Phys. Chem. Chem. Phys. 10(24), 3487-3497 (2008).
[CrossRef] [PubMed]

G. Meacci, J. Ries, E. Fischer-Friedrich, N. Kahya, P. Schwille, and K. Kruse, "Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy," Phys. Biol. 3(4), 255-263 (2006).
[CrossRef]

Scholpp, S.

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

Schwille, P.

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

J. Ries and P. Schwille, "New Concepts for Fluorescence Correlation Spectroscopy on Membranes," Phys. Chem. Chem. Phys. 10(24), 3487-3497 (2008).
[CrossRef] [PubMed]

S. Kim, K. Heinze, and P. Schwille, "Fluorescence correlation spectroscopy in living cells," Nat. Methods 4(11), 963-974 (2007).
[CrossRef] [PubMed]

G. Meacci, J. Ries, E. Fischer-Friedrich, N. Kahya, P. Schwille, and K. Kruse, "Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy," Phys. Biol. 3(4), 255-263 (2006).
[CrossRef]

K. Bacia, S. Kim, and P. Schwille, "Fluorescence cross-correlation spectroscopy in living cells," Nat. Methods 3(2), 83-89 (2006).
[CrossRef] [PubMed]

K. Bacia and P. Schwille, "A dynamic view of cellular processes by in vivo fluorescence auto-and crosscorrelation spectroscopy," Methods 29(1), 74-85 (2003).
[CrossRef] [PubMed]

Siegal-Gaskins, D.

C. C. Guet, L. Bruneaux, T. L. Min, D. Siegal-Gaskins, I. Figueroa, T. Emonet, and P. Cluzel, "Minimally invasive determination of mRNA concentration in single living bacteria," Nucleic Acids Res. 36(12), e73 (2008).
[CrossRef] [PubMed]

Solimena, M.

M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
[CrossRef] [PubMed]

Tcherniak, A.

A. Tcherniak, C. Reznik, S. Link, and C. F. Landes, "Fluorescence correlation spectroscopy: criteria for analysis in complex systems," Anal. Chem. 81(2), 746-754 (2009).
[CrossRef]

Trajkovski, M.

M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
[CrossRef] [PubMed]

von der Hocht, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements," ChemPhysChem 8(3), 433-443 (2007).
[CrossRef] [PubMed]

Wollheim, C. B.

M. Asfari, D. Janjic, P. Meda, G. Li, P. A. Halban, and C. B. Wollheim, "Establishment of 2-mercaptoethanoldependent differentiated insulin-secreting cell lines," Endocrinology 130(1), 167-178 (1992).
[CrossRef] [PubMed]

Yu, S. R.

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

Anal. Chem.

A. Tcherniak, C. Reznik, S. Link, and C. F. Landes, "Fluorescence correlation spectroscopy: criteria for analysis in complex systems," Anal. Chem. 81(2), 746-754 (2009).
[CrossRef]

Appl. Opt.

Biopolymers

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13 (1), 1-27 (1974).
[CrossRef]

ChemPhysChem

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements," ChemPhysChem 8(3), 433-443 (2007).
[CrossRef] [PubMed]

Endocrinology

M. Asfari, D. Janjic, P. Meda, G. Li, P. A. Halban, and C. B. Wollheim, "Establishment of 2-mercaptoethanoldependent differentiated insulin-secreting cell lines," Endocrinology 130(1), 167-178 (1992).
[CrossRef] [PubMed]

J. Cell. Biol.

M. Trajkovski, H. Mziaut, A. Altkruger, J. Ouwendijk, K. P. Knoch, S. Muller, and M. Solimena, "Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in beta-cells," J. Cell. Biol. 167(6), 1063-1074 (2004).
[CrossRef] [PubMed]

Methods

K. Bacia and P. Schwille, "A dynamic view of cellular processes by in vivo fluorescence auto-and crosscorrelation spectroscopy," Methods 29(1), 74-85 (2003).
[CrossRef] [PubMed]

Nat. Methods

S. Kim, K. Heinze, and P. Schwille, "Fluorescence correlation spectroscopy in living cells," Nat. Methods 4(11), 963-974 (2007).
[CrossRef] [PubMed]

K. Bacia, S. Kim, and P. Schwille, "Fluorescence cross-correlation spectroscopy in living cells," Nat. Methods 3(2), 83-89 (2006).
[CrossRef] [PubMed]

Nature

S. R. Yu,M. Burkhardt, M. Nowak, J. Ries, Z. Petr’asek, S. Scholpp, P. Schwille, and M. Brand, "Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules," Nature 461(7263), 533-536 (2009).
[CrossRef] [PubMed]

Nucleic Acids Res.

C. C. Guet, L. Bruneaux, T. L. Min, D. Siegal-Gaskins, I. Figueroa, T. Emonet, and P. Cluzel, "Minimally invasive determination of mRNA concentration in single living bacteria," Nucleic Acids Res. 36(12), e73 (2008).
[CrossRef] [PubMed]

Phys. Biol.

G. Meacci, J. Ries, E. Fischer-Friedrich, N. Kahya, P. Schwille, and K. Kruse, "Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy," Phys. Biol. 3(4), 255-263 (2006).
[CrossRef]

Phys. Chem. Chem. Phys.

J. Ries and P. Schwille, "New Concepts for Fluorescence Correlation Spectroscopy on Membranes," Phys. Chem. Chem. Phys. 10(24), 3487-3497 (2008).
[CrossRef] [PubMed]

Other

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

E. P. Petrov and P. Schwille, State of the art and novel trends in fluorescence correlation spectroscopy, in: Standardization in Fluorometry: State of the Art and Future Challenges, (Springer, Berlin Heidelberg New York, 2007).
[PubMed]

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Figures (2)

Fig. 1.
Fig. 1.

FCS measurements in complex systems. a: Image of ICA512-EGFP in Ins-1 cells. Bright secretory granules are clearly visible. b: Fluorescence intensity measured on ICA512-EGFP. Bright granules cause spikes in the fluorescence intensity and bleaching of an auto-fluorescent background or depletion of fluorophores lead to a slow change in the intensity over time. c: As a result, the auto-correlation curve is severely distorted (◇). Even a two-component fit (—) cannot accurately resolve the fast diffusion of the single proteins (τD = 1.0 ms, N = 4.2). A common way to reduce the distortions is to acquire several short correlation curves and discard distorted curves by hand-selection. The average of the best two curves calculated on 10 s long intervals of the intensity (◦) resembles the control measurement (cell without bright vesicles, amplitude normalized for visualization) much better, albeit with larger noise on the curve. d: 10 curves of 10 s. Obviously distorted curves (⋯) are discarded and the average of good curves (—) is used for further analysis (N = 10.5, τD = 0.73 ms). The inclusion of ambiguous curves (–.–) changes the result of the fit (N = 13.0, τD = 0.99 ms).

Fig. 2.
Fig. 2.

a,b: FCS on Streptavidin-Atto565 with 100 nm fluorescent beads. The beads cause spikes in the fluorescence intensity (a), leading to a distorted correlation curve (b,▫, fit: τ D1 = 0.09 ms). The average of many correlation curves calculated with short 1 s time intervals is less distorted (◊), but the fit values (τ D1 = 0.10 ms) still differ significantly from the control measurement consisting only of Streptavidin-Atto565 (+, τ D1 =0.185 ms). The automated selection algorithm discards the parts of the intensity trace with spikes (blue in a), the corresponding correlation curve (◦, τ D1 = 0.185 ms) is hardly distinguishable from the control (amplitude normalized for illustration). c,d: FCS on ICA512-EGFP in Ins-1 cells. The ICA512-EGFP partitions partially into secretory granules which cause spikes in the fluorescence intensity (c). In addition, bleaching of the fluorescent background and depletion of ICA512-EGFP due to photobleaching results in a decay of the average intensity. d: The correlation curves, calculated on 10 s long intensity traces (▫) and with short 2 s parts of the intensity trace (◊), are severely affected. The automated selection algorithm greatly reduces distortions (◦), although some residual slow component is visible when compared to the control (+), measured on ICA512-EGFP in cells showing only very few secretory granules and amplitude normalized to the corrected curve. e-h: Dependence of fit parameters on dG max: Curves were ordered according to their deviation from the other curves dGm and a fraction of the curves with the smallest dGm was averaged. Results from the fit of these average curves (e: number of particles N normalized for illustration, f: diffusion time of the fast component τ D1 and g: fast fraction F) and the maximum dGm (h) were plotted in dependence on the fraction of curves used for the average.

Equations (4)

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G ( τ ) = δI ( t ) δI ( t + τ ) I ( t ) 2
G ( τ ) = 1 N ( 1 + τ τ D ) 1 ( 1 + τ S 2 τ D ) 1 / 2 = 1 N G D ( τ )
G ( τ ) = 1 N ( 1 + T 1 T e τ / τ t ) ( F G D 1 ( τ ) + ( 1 F ) G D 2 ( τ ) )
d G k : = ( G k ( τ i ) G j ( τ i ) j k ) 2 i

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