Abstract

We describe the effect of optical aberrations on fluorescence fluctuations microscopy (FFM), when focusing through a single living cell. FFM measurements are performed in an aqueous fluorescent solution and prove to be a highly sensitive tool to assess the optical aberrations introduced by the cell. We demonstrate an adaptive optics (AO) system to remove the aberration-related bias in the FFM measurements. Our data show that AO is not only useful when imaging deep in tissues but also when performing FFM measurements through a single cellular layer. This work paves the way for the application of FFM to complex three-dimensional multicellular samples.

© 2013 Optical Society of America

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2012 (1)

2011 (2)

M. A. Digman and E. Gratton, Annu. Rev. Phys. Chem. 62, 645 (2011).
[CrossRef]

C. Leroux, I. Wang, J. Derouard, and A. Delon, Opt. Express 19, 26839 (2011).
[CrossRef]

2009 (1)

2008 (1)

Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, J. Microsc. 232, 343 (2008).
[CrossRef]

2007 (1)

E. Haustein and P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007).
[CrossRef]

2005 (2)

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, Biophys. J. 89, 1317 (2005).
[CrossRef]

M. A. Digman, P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz, and E. Gratton, Biophys. J. 88, L33 (2005).
[CrossRef]

2004 (2)

E. L. Elson, J. Biomed. Opt. 9, 857 (2004).
[CrossRef]

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, Curr. Pharm. Biotechnol. 5, 155 (2004).
[CrossRef]

2002 (2)

S. T. Hess and W. W. W. Webb, Biophys. J. 83, 2300 (2002).
[CrossRef]

M. Booth, M. Neil, and T. Wilson, J. Opt. Soc. Am. A 19, 2112 (2002).
[CrossRef]

1974 (1)

D. E. Koppel, Phys. Rev. A 10, 1938 (1974).
[CrossRef]

Beaurepaire, E.

Booth, M.

Botcherby, E.

Brown, C. M.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, Biophys. J. 89, 1317 (2005).
[CrossRef]

M. A. Digman, P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz, and E. Gratton, Biophys. J. 88, L33 (2005).
[CrossRef]

Buschmann, V.

Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, J. Microsc. 232, 343 (2008).
[CrossRef]

Débarre, D.

Delon, A.

Derouard, J.

Digman, M. A.

M. A. Digman and E. Gratton, Annu. Rev. Phys. Chem. 62, 645 (2011).
[CrossRef]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, Biophys. J. 89, 1317 (2005).
[CrossRef]

M. A. Digman, P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz, and E. Gratton, Biophys. J. 88, L33 (2005).
[CrossRef]

Elson, E. L.

E. L. Elson, J. Biomed. Opt. 9, 857 (2004).
[CrossRef]

Enderlein, J.

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, Curr. Pharm. Biotechnol. 5, 155 (2004).
[CrossRef]

Erdmann, R.

Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, J. Microsc. 232, 343 (2008).
[CrossRef]

Fitter, J.

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, Curr. Pharm. Biotechnol. 5, 155 (2004).
[CrossRef]

Gratton, E.

M. A. Digman and E. Gratton, Annu. Rev. Phys. Chem. 62, 645 (2011).
[CrossRef]

M. A. Digman, P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz, and E. Gratton, Biophys. J. 88, L33 (2005).
[CrossRef]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, Biophys. J. 89, 1317 (2005).
[CrossRef]

Gregor, I.

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, Curr. Pharm. Biotechnol. 5, 155 (2004).
[CrossRef]

Haustein, E.

E. Haustein and P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007).
[CrossRef]

Hess, S. T.

S. T. Hess and W. W. W. Webb, Biophys. J. 83, 2300 (2002).
[CrossRef]

Horwitz, A. R.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, Biophys. J. 89, 1317 (2005).
[CrossRef]

M. A. Digman, P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz, and E. Gratton, Biophys. J. 88, L33 (2005).
[CrossRef]

Koberling, F.

Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, J. Microsc. 232, 343 (2008).
[CrossRef]

Koppel, D. E.

D. E. Koppel, Phys. Rev. A 10, 1938 (1974).
[CrossRef]

Krämer, B.

Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, J. Microsc. 232, 343 (2008).
[CrossRef]

Leroux, C.

Macdonald, R.

Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, J. Microsc. 232, 343 (2008).
[CrossRef]

Mahou, P.

Neil, M.

Patra, D.

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, Curr. Pharm. Biotechnol. 5, 155 (2004).
[CrossRef]

Rüttinger,

Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, J. Microsc. 232, 343 (2008).
[CrossRef]

Schanne-Klein, M.

Schwille, P.

E. Haustein and P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007).
[CrossRef]

Sengupta, P.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, Biophys. J. 89, 1317 (2005).
[CrossRef]

M. A. Digman, P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz, and E. Gratton, Biophys. J. 88, L33 (2005).
[CrossRef]

Srinivas, S.

Wang, I.

Watanabe, T.

Webb, W. W. W.

S. T. Hess and W. W. W. Webb, Biophys. J. 83, 2300 (2002).
[CrossRef]

Wilson, T.

Wiseman, P. W.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, Biophys. J. 89, 1317 (2005).
[CrossRef]

M. A. Digman, P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz, and E. Gratton, Biophys. J. 88, L33 (2005).
[CrossRef]

Zeng, J.

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

E. Haustein and P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007).
[CrossRef]

Annu. Rev. Phys. Chem. (1)

M. A. Digman and E. Gratton, Annu. Rev. Phys. Chem. 62, 645 (2011).
[CrossRef]

Biomed. Opt. Express (1)

Biophys. J. (3)

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, Biophys. J. 89, 1317 (2005).
[CrossRef]

M. A. Digman, P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz, and E. Gratton, Biophys. J. 88, L33 (2005).
[CrossRef]

S. T. Hess and W. W. W. Webb, Biophys. J. 83, 2300 (2002).
[CrossRef]

Curr. Pharm. Biotechnol. (1)

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, Curr. Pharm. Biotechnol. 5, 155 (2004).
[CrossRef]

J. Biomed. Opt. (1)

E. L. Elson, J. Biomed. Opt. 9, 857 (2004).
[CrossRef]

J. Microsc. (1)

Rüttinger, V. Buschmann, B. Krämer, R. Erdmann, R. Macdonald, and F. Koberling, J. Microsc. 232, 343 (2008).
[CrossRef]

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

Opt. Express (1)

Opt. Lett. (1)

Phys. Rev. A (1)

D. E. Koppel, Phys. Rev. A 10, 1938 (1974).
[CrossRef]

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Figures (3)

Fig. 1.
Fig. 1.

(a) Optical layout. DPSSL, 561 nm diode-pumped solid state laser (Cobolt); DM, 97-actuator deformable mirror (AlpAO); OBJ, 63×/1.2 water-immersed microscope objective (Zeiss); SM, 3 mm X/Y galvanometric mirrors (Cambridge Technology); APD, single-photon-counting avalanche photodiode (PerkinElmer); MF, 1×Airy multimode fiber; DF, 600 nm long-pass dichroic filter (Chroma); SHWFS, 32×32 Shack–Hartmann wavefront sensor (AlpAO); CAM, wide-field camera (ANDOR sCMOS Zyla); FM1, flip mirror for DM calibration; FM2, flip mirror for transmission microscopy. (b) Schematic of the experiment. The z=0 focus position corresponds to the apex of the cell. FFM measurements are carried out in a fluorescent solution (sulforhodamine-B at a 15 nM concentration).

Fig. 2.
Fig. 2.

(a), (b) Wide-field images of cell1/cell2 over a 50×50μm field of view, observed with transmission microscopy. FFM measurements are carried out at the center of the field of view. (c), (d) Number of molecules as a function of the focusing distance above the center of cell1/cell2 before (dotted line) and after (solid line) aberration correction. (e), (f) Photon count rate above the center of cell1/cell2 before (dotted line) and after (solid line) aberration correction.

Fig. 3.
Fig. 3.

N˜(x,y) maps of the number of molecules over a 50×50μm field of view using the RICS technique. (a), (c) Cell1 without/with AO and (b), (d) Cell2 without and with AO.

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