Abstract

Fluorescence Correlation Spectroscopy (FCS) yields measurement parameters (number of molecules, diffusion time) that characterize the concentration and kinetics of fluorescent molecules within a supposedly known observation volume. Absolute derivation of concentrations and diffusion constants therefore requires preliminary calibrations of the confocal Point Spread Function with phantom solutions under perfectly controlled environmental conditions. In this paper, we quantify the influence of optical aberrations on single photon FCS and demonstrate a simple Adaptive Optics system for aberration correction. Optical aberrations are gradually introduced by focussing the excitation laser beam at increasing depths in fluorescent solutions with various refractive indices, which leads to drastic depth-dependent bias in the estimated FCS parameters. Aberration correction with a Deformable Mirror stabilizes these parameters within a range of several tens of μm into the solution. We also demonstrate, both theoretically and experimentally, that the molecular brightness scales as the Strehl ratio squared.

© 2011 OSA

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    [CrossRef] [PubMed]
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2011 (4)

M. A. Digman and E. Gratton, “Lessons in fluctuation correlation spectroscopy,” Annu. Rev. Phys. Chem.  62, 645–668 (2011).
[CrossRef] [PubMed]

O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
[CrossRef] [PubMed]

X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett.  36, 1062–1064 (2011).
[CrossRef] [PubMed]

X. Tao, O. Azucena, M. Fu, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars,” Opt. Lett.  36, 3389–3391 (2011).
[CrossRef] [PubMed]

2010 (1)

2009 (3)

C. B. Müller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, “Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding,” Soft Matter 5, 1358–1366 (2009).
[CrossRef]

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 1–13 (2009).
[CrossRef]

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, and H. Rigneault, “A versatile dual spot laser scanning confocal microscopy system for advanced fuorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum.  80, 083702 (2009).
[CrossRef] [PubMed]

2008 (2)

S. Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, “Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy,” J. Microsc.  232, 343–352 (2008).
[CrossRef] [PubMed]

T. Dertinger, A. Loman, B. Ewers, C. Müller, B. Krämer, and J. Enderlein, “The optics and performance of dual-focus fluorescence correlation spectroscopy,” Opt. Express 16, 14353–14368 (2008).
[CrossRef] [PubMed]

2007 (1)

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct.  36, 151–169 (2007).
[CrossRef] [PubMed]

2004 (4)

E. L. Elson, “Quick tour of fluorescence correlation spectroscopy,” J. Biomed. Opt.  9, 857–864 (2004).
[CrossRef] [PubMed]

J. D. Müller, “Cumulant analysis in fluorescence fluctuation spectroscopy,” Biophys. J.  86, 3981–3992 (2004).
[CrossRef] [PubMed]

B. Huang, T. D. Perroud, and R. N. Zare, “Photon counting histogram: one-photon excitation,” ChemPhysChem 5, 1523–1531 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. Booth, and T. Wilson, “Characterizing specimen induced aberrations for high NA adaptive optical microscopy,” Opt. Express 12, 6540–6552 (2004).
[CrossRef] [PubMed]

2002 (2)

M. Booth, M. Neil, and T. Wilson, “New modal wave-front sensor: application to adaptive confocal fluorescence microscopy and two-photon excitation fluorescence microscopy,” J. Opt. Soc. Am. A 19, 2112–2120 (2002).
[CrossRef]

S. T. Hess and W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys J.  83, 2300–2317 (2002).
[CrossRef] [PubMed]

2000 (1)

M. Neil, M. Booth, and T. Wilson, “Closed-loop aberration correction by use of a modal Zernike wave-front sensor,” Opt. Lett.  25, 1083–1085 (2000).
[CrossRef]

1998 (2)

M. Booth, M. Neil, and T. Wilson, “Aberration correction for confocal imaging in refractive-index-mismatched media,” J. Microsc.  192, 90–98 (1998).
[CrossRef]

J. Mertz, “Molecular photodynamics involved in multi-photon excitation fluorescence microscopy,” Eur. Phys. J. D 3, 53–66 (1998).
[CrossRef]

1995 (1)

J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem.  99, 13368–13379 (1995).
[CrossRef]

Aillaud, A.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, and H. Rigneault, “A versatile dual spot laser scanning confocal microscopy system for advanced fuorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum.  80, 083702 (2009).
[CrossRef] [PubMed]

Azucena, O.

X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett.  36, 1062–1064 (2011).
[CrossRef] [PubMed]

O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
[CrossRef] [PubMed]

X. Tao, O. Azucena, M. Fu, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars,” Opt. Lett.  36, 3389–3391 (2011).
[CrossRef] [PubMed]

O. Azucena, J. Crest, J. Cao, W. Sullivan, P. Kner, D. Gavel, D. Dillon, S. Olivier, and J. Kubby, “Wavefront aberration measurements and corrections through thick tissue using fluorescent microsphere reference beacons,” Opt. Express 18, 17521–17532 (2010).
[CrossRef] [PubMed]

Booth, M.

M. Schwertner, M. Booth, and T. Wilson, “Characterizing specimen induced aberrations for high NA adaptive optical microscopy,” Opt. Express 12, 6540–6552 (2004).
[CrossRef] [PubMed]

M. Booth, M. Neil, and T. Wilson, “New modal wave-front sensor: application to adaptive confocal fluorescence microscopy and two-photon excitation fluorescence microscopy,” J. Opt. Soc. Am. A 19, 2112–2120 (2002).
[CrossRef]

M. Neil, M. Booth, and T. Wilson, “Closed-loop aberration correction by use of a modal Zernike wave-front sensor,” Opt. Lett.  25, 1083–1085 (2000).
[CrossRef]

M. Booth, M. Neil, and T. Wilson, “Aberration correction for confocal imaging in refractive-index-mismatched media,” J. Microsc.  192, 90–98 (1998).
[CrossRef]

M. Booth, A. Kubasik-Thayil, A. Jesacher, D. Débarre, K. Grieve, and T. Wilson, “Adaptive optics in biomedical microscopy,” in Novel Techniques in Microscopy, OSA Technical Digest (CD) (Optical Society of America, 2009), paper NWA1.

Buschmann, V.

S. Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, “Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy,” J. Microsc.  232, 343–352 (2008).
[CrossRef] [PubMed]

Cao, J.

Chen, D.

X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett.  36, 1062–1064 (2011).
[CrossRef] [PubMed]

X. Tao, O. Azucena, M. Fu, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars,” Opt. Lett.  36, 3389–3391 (2011).
[CrossRef] [PubMed]

Crest, J.

O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
[CrossRef] [PubMed]

O. Azucena, J. Crest, J. Cao, W. Sullivan, P. Kner, D. Gavel, D. Dillon, S. Olivier, and J. Kubby, “Wavefront aberration measurements and corrections through thick tissue using fluorescent microsphere reference beacons,” Opt. Express 18, 17521–17532 (2010).
[CrossRef] [PubMed]

Débarre, D.

M. Booth, A. Kubasik-Thayil, A. Jesacher, D. Débarre, K. Grieve, and T. Wilson, “Adaptive optics in biomedical microscopy,” in Novel Techniques in Microscopy, OSA Technical Digest (CD) (Optical Society of America, 2009), paper NWA1.

Dertinger, T.

Digman, M. A.

M. A. Digman and E. Gratton, “Lessons in fluctuation correlation spectroscopy,” Annu. Rev. Phys. Chem.  62, 645–668 (2011).
[CrossRef] [PubMed]

Dillon, D.

Dorsey, N. E.

N. E. Dorsey, Properties of Ordinary Water-Substance in All Its Phases (New York, Reinhold Pub. Corp., 1940), p. 184.

Dross, N.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 1–13 (2009).
[CrossRef]

Eckert, T.

C. B. Müller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, “Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding,” Soft Matter 5, 1358–1366 (2009).
[CrossRef]

Elson, E. L.

E. L. Elson, “Quick tour of fluorescence correlation spectroscopy,” J. Biomed. Opt.  9, 857–864 (2004).
[CrossRef] [PubMed]

Enderlein, J.

C. B. Müller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, “Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding,” Soft Matter 5, 1358–1366 (2009).
[CrossRef]

T. Dertinger, A. Loman, B. Ewers, C. Müller, B. Krämer, and J. Enderlein, “The optics and performance of dual-focus fluorescence correlation spectroscopy,” Opt. Express 16, 14353–14368 (2008).
[CrossRef] [PubMed]

Erdmann, R.

S. Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, “Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy,” J. Microsc.  232, 343–352 (2008).
[CrossRef] [PubMed]

Ewers, B.

Fernandez, B.

X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett.  36, 1062–1064 (2011).
[CrossRef] [PubMed]

Ferrand, P.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, and H. Rigneault, “A versatile dual spot laser scanning confocal microscopy system for advanced fuorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum.  80, 083702 (2009).
[CrossRef] [PubMed]

Fu, M.

X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett.  36, 1062–1064 (2011).
[CrossRef] [PubMed]

X. Tao, O. Azucena, M. Fu, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars,” Opt. Lett.  36, 3389–3391 (2011).
[CrossRef] [PubMed]

Garcia, D.

X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett.  36, 1062–1064 (2011).
[CrossRef] [PubMed]

Gavel, D.

O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
[CrossRef] [PubMed]

O. Azucena, J. Crest, J. Cao, W. Sullivan, P. Kner, D. Gavel, D. Dillon, S. Olivier, and J. Kubby, “Wavefront aberration measurements and corrections through thick tissue using fluorescent microsphere reference beacons,” Opt. Express 18, 17521–17532 (2010).
[CrossRef] [PubMed]

Gratton, E.

M. A. Digman and E. Gratton, “Lessons in fluctuation correlation spectroscopy,” Annu. Rev. Phys. Chem.  62, 645–668 (2011).
[CrossRef] [PubMed]

Grieve, K.

M. Booth, A. Kubasik-Thayil, A. Jesacher, D. Débarre, K. Grieve, and T. Wilson, “Adaptive optics in biomedical microscopy,” in Novel Techniques in Microscopy, OSA Technical Digest (CD) (Optical Society of America, 2009), paper NWA1.

Haustein, E.

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct.  36, 151–169 (2007).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess and W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys J.  83, 2300–2317 (2002).
[CrossRef] [PubMed]

Huang, B.

B. Huang, T. D. Perroud, and R. N. Zare, “Photon counting histogram: one-photon excitation,” ChemPhysChem 5, 1523–1531 (2004).
[CrossRef] [PubMed]

Jesacher, A.

M. Booth, A. Kubasik-Thayil, A. Jesacher, D. Débarre, K. Grieve, and T. Wilson, “Adaptive optics in biomedical microscopy,” in Novel Techniques in Microscopy, OSA Technical Digest (CD) (Optical Society of America, 2009), paper NWA1.

Kner, P.

Koberling, F.

S. Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, “Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy,” J. Microsc.  232, 343–352 (2008).
[CrossRef] [PubMed]

Kotadia, S.

O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
[CrossRef] [PubMed]

Krämer, B.

S. Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, “Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy,” J. Microsc.  232, 343–352 (2008).
[CrossRef] [PubMed]

T. Dertinger, A. Loman, B. Ewers, C. Müller, B. Krämer, and J. Enderlein, “The optics and performance of dual-focus fluorescence correlation spectroscopy,” Opt. Express 16, 14353–14368 (2008).
[CrossRef] [PubMed]

Kress, A.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, and H. Rigneault, “A versatile dual spot laser scanning confocal microscopy system for advanced fuorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum.  80, 083702 (2009).
[CrossRef] [PubMed]

Kubasik-Thayil, A.

M. Booth, A. Kubasik-Thayil, A. Jesacher, D. Débarre, K. Grieve, and T. Wilson, “Adaptive optics in biomedical microscopy,” in Novel Techniques in Microscopy, OSA Technical Digest (CD) (Optical Society of America, 2009), paper NWA1.

Kubby, J.

X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett.  36, 1062–1064 (2011).
[CrossRef] [PubMed]

O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
[CrossRef] [PubMed]

X. Tao, O. Azucena, M. Fu, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars,” Opt. Lett.  36, 3389–3391 (2011).
[CrossRef] [PubMed]

O. Azucena, J. Crest, J. Cao, W. Sullivan, P. Kner, D. Gavel, D. Dillon, S. Olivier, and J. Kubby, “Wavefront aberration measurements and corrections through thick tissue using fluorescent microsphere reference beacons,” Opt. Express 18, 17521–17532 (2010).
[CrossRef] [PubMed]

Langowski, J.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 1–13 (2009).
[CrossRef]

Loman, A.

C. B. Müller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, “Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding,” Soft Matter 5, 1358–1366 (2009).
[CrossRef]

T. Dertinger, A. Loman, B. Ewers, C. Müller, B. Krämer, and J. Enderlein, “The optics and performance of dual-focus fluorescence correlation spectroscopy,” Opt. Express 16, 14353–14368 (2008).
[CrossRef] [PubMed]

Macdonald, R.

S. Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, “Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy,” J. Microsc.  232, 343–352 (2008).
[CrossRef] [PubMed]

Mertz, J.

J. Mertz, “Molecular photodynamics involved in multi-photon excitation fluorescence microscopy,” Eur. Phys. J. D 3, 53–66 (1998).
[CrossRef]

Mets, U.

J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem.  99, 13368–13379 (1995).
[CrossRef]

Müller, C.

Müller, C. B.

C. B. Müller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, “Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding,” Soft Matter 5, 1358–1366 (2009).
[CrossRef]

Müller, G.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 1–13 (2009).
[CrossRef]

Müller, J. D.

J. D. Müller, “Cumulant analysis in fluorescence fluctuation spectroscopy,” Biophys. J.  86, 3981–3992 (2004).
[CrossRef] [PubMed]

Neil, M.

M. Booth, M. Neil, and T. Wilson, “New modal wave-front sensor: application to adaptive confocal fluorescence microscopy and two-photon excitation fluorescence microscopy,” J. Opt. Soc. Am. A 19, 2112–2120 (2002).
[CrossRef]

M. Neil, M. Booth, and T. Wilson, “Closed-loop aberration correction by use of a modal Zernike wave-front sensor,” Opt. Lett.  25, 1083–1085 (2000).
[CrossRef]

M. Booth, M. Neil, and T. Wilson, “Aberration correction for confocal imaging in refractive-index-mismatched media,” J. Microsc.  192, 90–98 (1998).
[CrossRef]

Olivier, S.

O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
[CrossRef] [PubMed]

O. Azucena, J. Crest, J. Cao, W. Sullivan, P. Kner, D. Gavel, D. Dillon, S. Olivier, and J. Kubby, “Wavefront aberration measurements and corrections through thick tissue using fluorescent microsphere reference beacons,” Opt. Express 18, 17521–17532 (2010).
[CrossRef] [PubMed]

Perroud, T. D.

B. Huang, T. D. Perroud, and R. N. Zare, “Photon counting histogram: one-photon excitation,” ChemPhysChem 5, 1523–1531 (2004).
[CrossRef] [PubMed]

Pianta, M.

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, and H. Rigneault, “A versatile dual spot laser scanning confocal microscopy system for advanced fuorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum.  80, 083702 (2009).
[CrossRef] [PubMed]

Reinig, M.

O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
[CrossRef] [PubMed]

Richtering, W.

C. B. Müller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, “Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding,” Soft Matter 5, 1358–1366 (2009).
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J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem.  99, 13368–13379 (1995).
[CrossRef]

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P. Ferrand, M. Pianta, A. Kress, A. Aillaud, and H. Rigneault, “A versatile dual spot laser scanning confocal microscopy system for advanced fuorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum.  80, 083702 (2009).
[CrossRef] [PubMed]

Rüttinger, S.

S. Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, “Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy,” J. Microsc.  232, 343–352 (2008).
[CrossRef] [PubMed]

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Schwille, P.

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct.  36, 151–169 (2007).
[CrossRef] [PubMed]

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N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 1–13 (2009).
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O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
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O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
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X. Tao, O. Azucena, M. Fu, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars,” Opt. Lett.  36, 3389–3391 (2011).
[CrossRef] [PubMed]

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N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 1–13 (2009).
[CrossRef]

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S. T. Hess and W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys J.  83, 2300–2317 (2002).
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J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem.  99, 13368–13379 (1995).
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M. Booth, A. Kubasik-Thayil, A. Jesacher, D. Débarre, K. Grieve, and T. Wilson, “Adaptive optics in biomedical microscopy,” in Novel Techniques in Microscopy, OSA Technical Digest (CD) (Optical Society of America, 2009), paper NWA1.

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B. Huang, T. D. Perroud, and R. N. Zare, “Photon counting histogram: one-photon excitation,” ChemPhysChem 5, 1523–1531 (2004).
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X. Tao, O. Azucena, M. Fu, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars,” Opt. Lett.  36, 3389–3391 (2011).
[CrossRef] [PubMed]

X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett.  36, 1062–1064 (2011).
[CrossRef] [PubMed]

Zwerger, M.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 1–13 (2009).
[CrossRef]

Annu. Rev. Biophys. Biomol. Struct (1)

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct.  36, 151–169 (2007).
[CrossRef] [PubMed]

Annu. Rev. Phys. Chem (1)

M. A. Digman and E. Gratton, “Lessons in fluctuation correlation spectroscopy,” Annu. Rev. Phys. Chem.  62, 645–668 (2011).
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Biophys J (1)

S. T. Hess and W. W. Webb, “Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy,” Biophys J.  83, 2300–2317 (2002).
[CrossRef] [PubMed]

Biophys. J (1)

J. D. Müller, “Cumulant analysis in fluorescence fluctuation spectroscopy,” Biophys. J.  86, 3981–3992 (2004).
[CrossRef] [PubMed]

ChemPhysChem (1)

B. Huang, T. D. Perroud, and R. N. Zare, “Photon counting histogram: one-photon excitation,” ChemPhysChem 5, 1523–1531 (2004).
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S. Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, “Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy,” J. Microsc.  232, 343–352 (2008).
[CrossRef] [PubMed]

M. Booth, M. Neil, and T. Wilson, “Aberration correction for confocal imaging in refractive-index-mismatched media,” J. Microsc.  192, 90–98 (1998).
[CrossRef]

J. Opt. Soc. Am. A (1)

J. Phys. Chem (1)

J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem.  99, 13368–13379 (1995).
[CrossRef]

Opt. Express (3)

Opt. Lett (4)

O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, and J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett.  36, 825–827 (2011).
[CrossRef] [PubMed]

X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett.  36, 1062–1064 (2011).
[CrossRef] [PubMed]

X. Tao, O. Azucena, M. Fu, Y. Zuo, D. Chen, and J. Kubby, “Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars,” Opt. Lett.  36, 3389–3391 (2011).
[CrossRef] [PubMed]

M. Neil, M. Booth, and T. Wilson, “Closed-loop aberration correction by use of a modal Zernike wave-front sensor,” Opt. Lett.  25, 1083–1085 (2000).
[CrossRef]

PLoS ONE (1)

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 1–13 (2009).
[CrossRef]

Rev. Sci. Instrum (1)

P. Ferrand, M. Pianta, A. Kress, A. Aillaud, and H. Rigneault, “A versatile dual spot laser scanning confocal microscopy system for advanced fuorescence correlation spectroscopy analysis in living cell,” Rev. Sci. Instrum.  80, 083702 (2009).
[CrossRef] [PubMed]

Soft Matter (1)

C. B. Müller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, “Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding,” Soft Matter 5, 1358–1366 (2009).
[CrossRef]

Other (4)

N. E. Dorsey, Properties of Ordinary Water-Substance in All Its Phases (New York, Reinhold Pub. Corp., 1940), p. 184.

D. R. Lide, ed., Handbook of Chemistry and Physics (CRC Press, Cleveland, 2006).

P. Kapusta, “Absolute diffusion coefficients: compilation of reference data for FCS calibration,” http://www.picoquant.com/technotes/appnote_diffusion_coefficients.pdf .

M. Booth, A. Kubasik-Thayil, A. Jesacher, D. Débarre, K. Grieve, and T. Wilson, “Adaptive optics in biomedical microscopy,” in Novel Techniques in Microscopy, OSA Technical Digest (CD) (Optical Society of America, 2009), paper NWA1.

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Figures (6)

Fig. 1
Fig. 1

Optical layout of the experiment; the flip mirror is removed while performing FCS measurements.

Fig. 2
Fig. 2

Calibration of the DM with a SHWFS. The amplitudes of the measured Zernike modes (solid lines), astigmatism (a4), coma (a6) and spherical aberration (a10) are plotted versus the targeted values (x-axis). The corresponding RMS residual errors ɛ4, ɛ6, ɛ10. are also shown (dash dot lines).

Fig. 3
Fig. 3

ACF recorded in the 70.4 % glycerol solutions, without (left) and with (right) AO. The amplitude of the ACF decreases dramatically with increasing observation depth (from 10 to 45 μm) when the AO is not switched on. The superimposed dark solid lines are the fits performed with Eq. (4).

Fig. 4
Fig. 4

Variations of the estimated FCS parameters normalized to their values at the reference depth z = 10 μm, N/N10 (red) and τDD10 (green), in the 70.4 % (open triangles) and 50 % (open circles) glycerol solutions, without (left) and with (right) AO.

Fig. 5
Fig. 5

Aberrations corrected by the DM in the glycerol solutions: spherical aberration amplitudes (a10, open circles) and the residual aberrations (r10, open squares) for the 50% glycerol solution (blue) and for the 70.4% one (red); solid lines are linear fits of the spherical aberration amplitudes.

Fig. 6
Fig. 6

Comparison of the molecular brightness with the Strehl ratio squared, computed using Eq. (8) (solid lines). Left graph: the ordinate on the vertical axis, CRM/CRM10, is normalized to its value at the reference depth z = 10 μm, and the horizontal axis is the overall RMS amplitude of the corrected aberrations, σwf, in the glycerol solutions (50 % solution in blue and 70.4 % one in red). Right graph: the ordinate on the vertical axis, CRM/CRMa=0 is normalized to its value when no single mode aberration is applied, the horizontal axis is the amplitude of a single Zernike mode (a4: astigmatism in magenta, a10: spherical aberration in green), generated by the DM in an A647 80 nM pure water solution, while other aberrations are corrected.

Equations (9)

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

G ( τ ) = I ( t ) I ( t + τ ) I ( t ) 2
N = C × π 3 / 2 w r 2 w z
τ D = w r 2 4 D
G ( τ ) = 1 + 1 N ( 1 + f T 1 f T e τ / τ T ) ( 1 + τ τ D ) 1 ( 1 + τ S 2 τ D ) 1 / 2
V fcs = [ PSF con d r ] 2 PSF con 2 d r
CR = η × C × S t r 2 PSF con d r
CRM = η 2 3 / 2 S t r 2
S t r exp ( 2 π × σ w f λ ) 2
σ w f = i = 4 i = 10 a i 2

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