s . bi o - ph ] 1 7 O ct 2 01 1 Adaptive Optics for Fluorescence Correlation Spectroscopy

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 Optical Society of America OCIS codes: (000.0000) General. References and links 1. M. A. Digman and E. Gratton, “Lessons in Fluctuation Correlation Spectroscopy,” Annu. Rev. Phys. Chem. 62, 645–668 (2011). 2. E. Haustein and P. Schwille, “Fluorescence Correlation Spectroscopy: Novel Variations of an Established Technique,”, Annu. Rev. Biophys. Biomol. Struct. 36, 151–169 (2007). 3. E. L. Elson, “Quick tour of fluorescence correlation spectroscopy,” J. Biomed. Opt. 9, 857–864 (2004). 4. S.T. Hess and W.W. Webb, “Focal Volume Optics and Experimental Artifacts in Confocal Fluorescence Correlation Spectroscopy,” Biophys J. 83, 2300–2317 (2002). 5. J. D. Müller, “Cumulant Analysis in Fluorescence Fluctuation Spectroscopy,” Biophys. J. 86, 3981–3992 (2004). 6. B. Huang, T. D. Perroud, and R. N. Zare, ”Photon Counting Histogram: One-Photon Excitation,” ChemPhysChem 5, 1523–1531(2004). 7. 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). 8. T. Dertinger, A. Loman, B. Ewers, C. Mller, B. Kräamer, and J. Enderlein, “The optics and performance of dual-focus fluorescence correlation spectroscopy,” Opt. Express 16, 14353-14368 (2008). 9. C. B. Müller, T. Eckert, A. Loman, J. Enderlein and W. Richtering, “Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding,” SoftMatter 5, 1358-1366 (2009). 10. N. Dross, C. Spriet, M. Zwerger, G. Mller, W. Waldeck, J. Langowski, “Mapping eGFP Oligomer Mobility in Living Cell Nuclei,” PLoS ONE 4, e5041 1–13(2009). 11. 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, 083702-1–8 (2009). 12. J. Widengren, Ülo Mets and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimetal Study,” J. Phys. Chem. 99, 13368–13379 (1995). 13. J. Mertz, “Molecular photodynamics involved in multi-photon excitation fluorescence microscopy,” Eur. Phys. J. D 3, 53–66 (1998). 14. M. Schwertner, M. Booth, and T. Wilson, “Characterizing specimen induced aberrations for high NA adaptive optical microscopy,” Opt. Express 12, 6540-6552 (2004). 15. M. Booth, A. Kubasik-Thayil, A. Jesacher, D. Debarre, 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. 16. 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). 17. 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). 18. 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). 19. 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). 20. 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). 21. 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). 22. N.E. Dorsey, Properties of Ordinary Water-Substance, New York (1940), p. 184 23. Handbook of Chemistry and Physics, D.R. Lide, ed. (CRC Press, Cleveland, 2006) 24. P. Kapusta, “Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration,” http://www.picoquant.com/technotes/appnote diffusion coefficients.pdf 25. M. Booth, M. Neil, and T. Wilson,“Aberration correction for confocal imaging in refractive-index-mismatched media,”J. Microsc. 192, 90–98 (1998).