Abstract

Deep imaging in turbid media such as biological tissue is challenging due to scattering and optical aberrations. Adaptive optics has the potential to compensate the tissue aberrations. We present a wavefront sensing scheme for multi-photon scanning microscopes using the pulsed, near-infrared light reflected back from the sample utilising coherence gating and a confocal pinhole to isolate the light from a layer of interest. By interfering the back-reflected light with a tilted reference beam, we create a fringe pattern with a known spatial carrier frequency in an image of the back-aperture plane of the microscope objective. The wavefront aberrations distort this fringe pattern and thereby imprint themselves at the carrier frequency, which allows us to separate the aberrations in the Fourier domain from low spatial frequency noise. A Fourier analysis of the modulated fringes combined with a virtual Shack-Hartmann sensor for smoothing yields a modal representation of the wavefront suitable for correction. We show results with this method correcting both DM-induced and sample-induced aberrations in rat tail collagen fibres as well as a Hoechst-stained MCF-7 spheroid of cancer cells.

© 2014 Optical Society of America

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  1. M. Minsky, “Microscopy apparatus,” US Patent 3,013,467 (1961).
  2. D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).
  3. J. B. Pawley, ed., Handbook Of Biological Confocal Microscopy, 3rd ed. (SpringerUS, Boston, MA, 2006).
    [CrossRef]
  4. W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
    [CrossRef] [PubMed]
  5. W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nature Biotechnol. 21, 1369–1377 (2003).
    [CrossRef]
  6. M. Schwertner, M. J. Booth, M. A. A. Neil, T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213, 11–19 (2004).
    [CrossRef]
  7. M. Schwertner, M. J. Booth, T. Wilson, “Specimen-induced distortions in light microscopy,” J. Microsc. 228, 97–102 (2007).
    [CrossRef] [PubMed]
  8. C. J. de Grauw, J. M. Vroom, H. T. M. van der Voort, H. C. Gerritsen, “Imaging Properties in Two-Photon Excitation Microscopy and Effects of Refractive-Index Mismatch in Thick Specimens,” Appl. Opt. 38, 5995–6003 (1999).
    [CrossRef]
  9. M. J. Booth, “Adaptive optics in microscopy,” Philos. Trans. R. Soc., A 365, 2829–2843 (2007).
    [CrossRef]
  10. M. J. Booth, “Wavefront sensorless adaptive optics for large aberrations,” Opt. Lett. 32, 5–7 (2007).
    [CrossRef]
  11. P. Marsh, D. Burns, J. Girkin, “Practical implementation of adaptive optics in multiphoton microscopy,” Opt. Express 11, 1123–1130 (2003).
    [CrossRef] [PubMed]
  12. D. Débarre, E. J. Botcherby, T. Watanabe, S. Srinivas, M. J. Booth, T. Wilson, “Image-based adaptive optics for two-photon microscopy,” Opt. Lett. 34, 2495–2497 (2009).
    [CrossRef] [PubMed]
  13. D. Débarre, M. J. Booth, T. Wilson, “Image based adaptive optics through optimisation of low spatial frequencies,” Opt. Express 15, 8176–8190 (2007).
    [CrossRef] [PubMed]
  14. A. Facomprez, E. Beaurepaire, D. Débarre, “Accuracy of correction in modal sensorless adaptive optics,” Opt. Express 20, 2598–2612 (2012).
    [CrossRef] [PubMed]
  15. O. Azucena, J. Crest, J. Cao, W. Sullivan, P. Kner, D. Gavel, D. Dillon, S. Olivier, J. Kubby, “Wavefront aberration measurements and corrections through thick tissue using fluorescent microsphere reference beacons,” Opt. Express 18, 17521–17532 (2010).
    [CrossRef] [PubMed]
  16. O. Azucena, J. Crest, S. Kotadia, W. Sullivan, X. Tao, M. Reinig, D. Gavel, S. Olivier, J. Kubby, “Adaptive optics wide-field microscopy using direct wavefront sensing,” Opt. Lett. 36, 825–827 (2011).
    [CrossRef] [PubMed]
  17. X. Tao, A. Norton, M. Kissel, O. Azucena, J. Kubby, “Adaptive optical two-photon microscopy using autofluorescent guide stars,” Opt. Lett. 38, 5075–5078 (2013).
    [CrossRef] [PubMed]
  18. X. Tao, J. Crest, S. Kotadia, O. Azucena, D. C. Chen, W. Sullivan, J. Kubby, “Live imaging using adaptive optics with fluorescent protein guide-stars,” Opt. Express 20, 15969, 2012).
    [CrossRef] [PubMed]
  19. X. Tao, O. Azucena, M. Fu, Y. Zuo, D. C. Chen, J. Kubby, “Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars,” Opt. Lett. 36, 3389–3391 (2011).
    [CrossRef] [PubMed]
  20. X. Tao, B. Fernandez, O. Azucena, M. Fu, D. Garcia, Y. Zuo, D. C. Chen, J. Kubby, “Adaptive optics confocal microscopy using direct wavefront sensing,” Opt. Lett. 36, 1062–1064 (2011).
    [CrossRef] [PubMed]
  21. R. Foy, A. Labeyrie, “Feasibility of adaptive telescope with laser probe,” Astron. Astrophys. 152, L29–L31 (1985).
  22. R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
    [CrossRef]
  23. M. Feierabend, M. Rückel, W. Denk, “Coherence-gated wave-front sensing in strongly scattering samples,” Opt. Lett. 29, 2255–2257 (2004).
    [CrossRef] [PubMed]
  24. M. Rückel, J. A. Mack-Bucher, W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Nat. Acad. Sci. USA 103, 17137–17142 (2006).
    [CrossRef]
  25. S. Tuohy, A. G. Podoleanu, “Depth-resolved wavefront aberrations using a coherence-gated Shack-Hartmann wavefront sensor,” Opt. Express 18, 3458–3476 (2010).
    [CrossRef] [PubMed]
  26. J. W. Cha, J. Ballesta, P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15, 046022, 2010).
    [CrossRef] [PubMed]
  27. D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
    [CrossRef] [PubMed]
  28. M. Takeda, H. Ina, S. Kobayashi, “Fourier-transform method of fringe-pattern analysis for computer-based topography and interferometry,” J. Opt. Soc. Am. 72, 156–160 (1982).
    [CrossRef]
  29. W. W. Macy, “Two-dimensional fringe-pattern analysis,” Appl. Opt. 22, 3898–3901 (1983).
    [CrossRef] [PubMed]
  30. D. J. Bone, H. A. Bachor, R. J. Sandeman, “Fringe-pattern analysis using a 2-D Fourier transform,” Appl. Opt. 25, 1653–1660 (1986).
    [CrossRef] [PubMed]
  31. M. Takeda, “Spatial-carrier fringe-pattern analysis and its applications to precision interferometry and profilometry: An overview,” Industrial Metrology 1, 79–99 (1990).
    [CrossRef]
  32. C. Paterson, I. Munro, J. C. Dainty, “A low cost adaptive optics system using a membrane mirror,” Opt. Express 6, 175, 2000).
    [CrossRef] [PubMed]
  33. S. A. Rahman, M. J. Booth, “Direct wavefront sensing in adaptive optical microscopy using backscattered light,” Appl. Opt. 52, 5523–5532 (2013).
    [CrossRef] [PubMed]
  34. J. Wang, J.-F. Léger, J. Binding, A. C. Boccara, S. Gigan, L. Bourdieu, “Measuring aberrations in the rat brain by coherence-gated wavefront sensing using a Linnik interferometer,” Biomed. Opt. Express 3, 2510–2525 (2012).
    [CrossRef] [PubMed]
  35. M. Feierabend, “Coherence-Gated Wave-Front Sensing in Strongly Scattering Samples,” Ph.D. thesis, Ruperto-Carola University of Heidelberg, Heidelberg, Germany (2004).
  36. G. J. Brakenhoff, K. Visscher, H. T. M. Voort, Size and Shape of The Confocal Spot: Control and Relation to 3D Imaging and Image Processing, in Handbook of Biological Confocal Microscopy” J. B. Pawley, ed. (Plenum Press, New York, USA, 1990), pp. 87–91, revised ed.
    [CrossRef]
  37. J. A. Nelder, R. Mead, “A Simplex Method for Function Minimization,” The Computer Journal 7, 308–313 (1965).
    [CrossRef]
  38. J. Antonello, M. Verhaegen, R. Fraanje, T. van Werkhoven, H. C. Gerritsen, C. U. Keller, “Semidefinite programming for model-based sensorless adaptive optics,” J. Opt. Soc. Am. A 29, 2428–2438 (2012).
    [CrossRef]
  39. P. T. C. So, C. Y. Dong, B. R. Masters, K. M. Berland, “Two-Photon Excitation Fluorescence Microscopy,” Annual Review of Biomedical Engineering 2, 399–429 (2000).
    [CrossRef]
  40. M. Peck, Interferometry mathematics, algorithms and data(2010).
  41. K. Itoh, “Analysis of the phase unwrapping algorithm,” Appl. Opt. 21, 2470, 1982).
    [CrossRef] [PubMed]
  42. D. C. Ghiglia, M. D. Pritt, Two-Dimensional Phase Unwrapping: Theory, Algorithms, and Software, 1st ed. (Wiley-Interscience, 1998).
  43. W. H. Southwell, “Wave-front estimation from wave-front slope measurements,” J. Opt. Soc. Am. 70, 998–1006 (1980).
    [CrossRef]
  44. F. von Zernike, “Beugungstheorie des schneidenver-fahrens und seiner verbesserten form, der phasenkontrastmethode,” Physica 1, 689–704 (1934).
    [CrossRef]
  45. R. J. Noll, “Zernike polynomials and atmospheric turbulence,” J. Opt. Soc. Am. 66, 207–211 (1976).
    [CrossRef]
  46. J. W. Goodman, “Some fundamental properties of speckle,” J. Opt. Soc. Am. 66, 1145–1150 (1976).
    [CrossRef]
  47. P. Artal, S. Marcos, R. Navarro, D. R. Williams, “Odd aberrations and double-pass measurements of retinal image quality,” J. Opt. Soc. Am. A 12, 195–201 (1995).
    [CrossRef]
  48. P. Artal, I. Iglesias, N. López-Gil, D. G. Green, “Double-pass measurements of the retinal-image quality with unequal entrance and exit pupil sizes and the reversibility of the eye’s optical system,” J. Opt. Soc. Am. A 12, 2358–2366 (1995).
    [CrossRef]
  49. M. Rückel, W. Denk, “Properties of coherence-gated wavefront sensing,” J. Opt. Soc. Am. A 24, 3517–3529 (2007).
    [CrossRef]
  50. H. D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, “A human cell line from a pleural effusion derived from a breast carcinoma.” J. Natl. Cancer Inst. 51, 1409–1416 (1973).
    [PubMed]
  51. J. Friedrich, C. Seidel, R. Ebner, L. A. Kunz-Schughart, “Spheroid-based drug screen: considerations and practical approach,” Nature Protocols 4, 309–324 (2009).
    [CrossRef] [PubMed]
  52. R. Fiolka, K. Si, M. Cui, “Complex wavefront corrections for deep tissue focusing using low coherence backscattered light,” Opt. Express 20, 16532–16543 (2012).
    [CrossRef]
  53. J. Zeng, P. Mahou, M.-C. Schanne-Klein, E. Beaurepaire, D. Débarre, “3D resolved mapping of optical aberrations in thick tissues,” Biomedical Opt. Express 3, 1898–1913 (2012).
    [CrossRef]

2013 (2)

2012 (6)

2011 (3)

2010 (3)

2009 (2)

J. Friedrich, C. Seidel, R. Ebner, L. A. Kunz-Schughart, “Spheroid-based drug screen: considerations and practical approach,” Nature Protocols 4, 309–324 (2009).
[CrossRef] [PubMed]

D. Débarre, E. J. Botcherby, T. Watanabe, S. Srinivas, M. J. Booth, T. Wilson, “Image-based adaptive optics for two-photon microscopy,” Opt. Lett. 34, 2495–2497 (2009).
[CrossRef] [PubMed]

2007 (5)

2006 (1)

M. Rückel, J. A. Mack-Bucher, W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Nat. Acad. Sci. USA 103, 17137–17142 (2006).
[CrossRef]

2004 (2)

M. Schwertner, M. J. Booth, M. A. A. Neil, T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213, 11–19 (2004).
[CrossRef]

M. Feierabend, M. Rückel, W. Denk, “Coherence-gated wave-front sensing in strongly scattering samples,” Opt. Lett. 29, 2255–2257 (2004).
[CrossRef] [PubMed]

2003 (2)

P. Marsh, D. Burns, J. Girkin, “Practical implementation of adaptive optics in multiphoton microscopy,” Opt. Express 11, 1123–1130 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nature Biotechnol. 21, 1369–1377 (2003).
[CrossRef]

2000 (2)

P. T. C. So, C. Y. Dong, B. R. Masters, K. M. Berland, “Two-Photon Excitation Fluorescence Microscopy,” Annual Review of Biomedical Engineering 2, 399–429 (2000).
[CrossRef]

C. Paterson, I. Munro, J. C. Dainty, “A low cost adaptive optics system using a membrane mirror,” Opt. Express 6, 175, 2000).
[CrossRef] [PubMed]

1999 (1)

1995 (2)

1991 (2)

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

1990 (2)

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

M. Takeda, “Spatial-carrier fringe-pattern analysis and its applications to precision interferometry and profilometry: An overview,” Industrial Metrology 1, 79–99 (1990).
[CrossRef]

1989 (1)

D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).

1986 (1)

1985 (1)

R. Foy, A. Labeyrie, “Feasibility of adaptive telescope with laser probe,” Astron. Astrophys. 152, L29–L31 (1985).

1983 (1)

1982 (2)

1980 (1)

1976 (2)

1973 (1)

H. D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, “A human cell line from a pleural effusion derived from a breast carcinoma.” J. Natl. Cancer Inst. 51, 1409–1416 (1973).
[PubMed]

1965 (1)

J. A. Nelder, R. Mead, “A Simplex Method for Function Minimization,” The Computer Journal 7, 308–313 (1965).
[CrossRef]

1934 (1)

F. von Zernike, “Beugungstheorie des schneidenver-fahrens und seiner verbesserten form, der phasenkontrastmethode,” Physica 1, 689–704 (1934).
[CrossRef]

Albert, S.

H. D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, “A human cell line from a pleural effusion derived from a breast carcinoma.” J. Natl. Cancer Inst. 51, 1409–1416 (1973).
[PubMed]

Ameer, G. A.

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

Antonello, J.

Artal, P.

Azucena, O.

Bachor, H. A.

Ballesta, J.

J. W. Cha, J. Ballesta, P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15, 046022, 2010).
[CrossRef] [PubMed]

Beaurepaire, E.

A. Facomprez, E. Beaurepaire, D. Débarre, “Accuracy of correction in modal sensorless adaptive optics,” Opt. Express 20, 2598–2612 (2012).
[CrossRef] [PubMed]

J. Zeng, P. Mahou, M.-C. Schanne-Klein, E. Beaurepaire, D. Débarre, “3D resolved mapping of optical aberrations in thick tissues,” Biomedical Opt. Express 3, 1898–1913 (2012).
[CrossRef]

Berland, K. M.

P. T. C. So, C. Y. Dong, B. R. Masters, K. M. Berland, “Two-Photon Excitation Fluorescence Microscopy,” Annual Review of Biomedical Engineering 2, 399–429 (2000).
[CrossRef]

Binding, J.

Boccara, A. C.

Boeke, B. R.

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

Bone, D. J.

Booth, M. J.

Botcherby, E. J.

Bourdieu, L.

Brakenhoff, G. J.

G. J. Brakenhoff, K. Visscher, H. T. M. Voort, Size and Shape of The Confocal Spot: Control and Relation to 3D Imaging and Image Processing, in Handbook of Biological Confocal Microscopy” J. B. Pawley, ed. (Plenum Press, New York, USA, 1990), pp. 87–91, revised ed.
[CrossRef]

Brennan, M.

H. D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, “A human cell line from a pleural effusion derived from a breast carcinoma.” J. Natl. Cancer Inst. 51, 1409–1416 (1973).
[PubMed]

Browne, S. L.

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

Burns, D.

Cao, J.

Cha, J. W.

J. W. Cha, J. Ballesta, P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15, 046022, 2010).
[CrossRef] [PubMed]

Chang, W.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Chen, D. C.

Crest, J.

Cui, M.

Dainty, J. C.

de Grauw, C. J.

Débarre, D.

Denk, W.

M. Rückel, W. Denk, “Properties of coherence-gated wavefront sensing,” J. Opt. Soc. Am. A 24, 3517–3529 (2007).
[CrossRef]

M. Rückel, J. A. Mack-Bucher, W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Nat. Acad. Sci. USA 103, 17137–17142 (2006).
[CrossRef]

M. Feierabend, M. Rückel, W. Denk, “Coherence-gated wave-front sensing in strongly scattering samples,” Opt. Lett. 29, 2255–2257 (2004).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Dillon, D.

Dong, C. Y.

P. T. C. So, C. Y. Dong, B. R. Masters, K. M. Berland, “Two-Photon Excitation Fluorescence Microscopy,” Annual Review of Biomedical Engineering 2, 399–429 (2000).
[CrossRef]

Ebner, R.

J. Friedrich, C. Seidel, R. Ebner, L. A. Kunz-Schughart, “Spheroid-based drug screen: considerations and practical approach,” Nature Protocols 4, 309–324 (2009).
[CrossRef] [PubMed]

Et, A.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Facomprez, A.

Feierabend, M.

M. Feierabend, M. Rückel, W. Denk, “Coherence-gated wave-front sensing in strongly scattering samples,” Opt. Lett. 29, 2255–2257 (2004).
[CrossRef] [PubMed]

M. Feierabend, “Coherence-Gated Wave-Front Sensing in Strongly Scattering Samples,” Ph.D. thesis, Ruperto-Carola University of Heidelberg, Heidelberg, Germany (2004).

Fernandez, B.

Fiolka, R.

Flotte, T.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Foy, R.

R. Foy, A. Labeyrie, “Feasibility of adaptive telescope with laser probe,” Astron. Astrophys. 152, L29–L31 (1985).

Fraanje, R.

Fried, D. L.

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

Friedrich, J.

J. Friedrich, C. Seidel, R. Ebner, L. A. Kunz-Schughart, “Spheroid-based drug screen: considerations and practical approach,” Nature Protocols 4, 309–324 (2009).
[CrossRef] [PubMed]

Fu, M.

Fugate, R. Q.

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

Garcia, D.

Gavel, D.

Gerritsen, H. C.

Ghiglia, D. C.

D. C. Ghiglia, M. D. Pritt, Two-Dimensional Phase Unwrapping: Theory, Algorithms, and Software, 1st ed. (Wiley-Interscience, 1998).

Gigan, S.

Girkin, J.

Goodman, J. W.

Green, D. G.

Gregory, K.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Hee, M. R.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Huang, D.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Iglesias, I.

Ina, H.

Itoh, K.

Keller, C. U.

Kissel, M.

Kner, P.

Kobayashi, S.

Kotadia, S.

Kubby, J.

Kunz-Schughart, L. A.

J. Friedrich, C. Seidel, R. Ebner, L. A. Kunz-Schughart, “Spheroid-based drug screen: considerations and practical approach,” Nature Protocols 4, 309–324 (2009).
[CrossRef] [PubMed]

Labeyrie, A.

R. Foy, A. Labeyrie, “Feasibility of adaptive telescope with laser probe,” Astron. Astrophys. 152, L29–L31 (1985).

Léger, J.-F.

Lin, C. P.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Long, A.

H. D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, “A human cell line from a pleural effusion derived from a breast carcinoma.” J. Natl. Cancer Inst. 51, 1409–1416 (1973).
[PubMed]

López-Gil, N.

Mack-Bucher, J. A.

M. Rückel, J. A. Mack-Bucher, W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Nat. Acad. Sci. USA 103, 17137–17142 (2006).
[CrossRef]

Macy, W. W.

Mahou, P.

J. Zeng, P. Mahou, M.-C. Schanne-Klein, E. Beaurepaire, D. Débarre, “3D resolved mapping of optical aberrations in thick tissues,” Biomedical Opt. Express 3, 1898–1913 (2012).
[CrossRef]

Marcos, S.

Marsh, P.

Masters, B. R.

P. T. C. So, C. Y. Dong, B. R. Masters, K. M. Berland, “Two-Photon Excitation Fluorescence Microscopy,” Annual Review of Biomedical Engineering 2, 399–429 (2000).
[CrossRef]

Mead, R.

J. A. Nelder, R. Mead, “A Simplex Method for Function Minimization,” The Computer Journal 7, 308–313 (1965).
[CrossRef]

Minsky, M.

M. Minsky, “Microscopy apparatus,” US Patent 3,013,467 (1961).

Munro, I.

Navarro, R.

Neil, M. A. A.

M. Schwertner, M. J. Booth, M. A. A. Neil, T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213, 11–19 (2004).
[CrossRef]

Nelder, J. A.

J. A. Nelder, R. Mead, “A Simplex Method for Function Minimization,” The Computer Journal 7, 308–313 (1965).
[CrossRef]

Noll, R. J.

Norton, A.

Olivier, S.

Paterson, C.

Peck, M.

M. Peck, Interferometry mathematics, algorithms and data(2010).

Podoleanu, A. G.

Pritt, M. D.

D. C. Ghiglia, M. D. Pritt, Two-Dimensional Phase Unwrapping: Theory, Algorithms, and Software, 1st ed. (Wiley-Interscience, 1998).

Puliafito, C. A.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Rahman, S. A.

Reinig, M.

Roberts, P. H.

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

Ruane, R. E.

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

Rückel, M.

Sandeman, R. J.

Schanne-Klein, M.-C.

J. Zeng, P. Mahou, M.-C. Schanne-Klein, E. Beaurepaire, D. Débarre, “3D resolved mapping of optical aberrations in thick tissues,” Biomedical Opt. Express 3, 1898–1913 (2012).
[CrossRef]

Schuman, J. S.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Schwertner, M.

M. Schwertner, M. J. Booth, T. Wilson, “Specimen-induced distortions in light microscopy,” J. Microsc. 228, 97–102 (2007).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. A. Neil, T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213, 11–19 (2004).
[CrossRef]

Seidel, C.

J. Friedrich, C. Seidel, R. Ebner, L. A. Kunz-Schughart, “Spheroid-based drug screen: considerations and practical approach,” Nature Protocols 4, 309–324 (2009).
[CrossRef] [PubMed]

Shotton, D. M.

D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).

Si, K.

So, P. T. C.

J. W. Cha, J. Ballesta, P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15, 046022, 2010).
[CrossRef] [PubMed]

P. T. C. So, C. Y. Dong, B. R. Masters, K. M. Berland, “Two-Photon Excitation Fluorescence Microscopy,” Annual Review of Biomedical Engineering 2, 399–429 (2000).
[CrossRef]

Soule, H. D.

H. D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, “A human cell line from a pleural effusion derived from a breast carcinoma.” J. Natl. Cancer Inst. 51, 1409–1416 (1973).
[PubMed]

Southwell, W. H.

Srinivas, S.

Stinson, W. G.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Sullivan, W.

Swanson, E. A.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

Takeda, M.

M. Takeda, “Spatial-carrier fringe-pattern analysis and its applications to precision interferometry and profilometry: An overview,” Industrial Metrology 1, 79–99 (1990).
[CrossRef]

M. Takeda, H. Ina, S. Kobayashi, “Fourier-transform method of fringe-pattern analysis for computer-based topography and interferometry,” J. Opt. Soc. Am. 72, 156–160 (1982).
[CrossRef]

Tao, X.

Tuohy, S.

Tyler, G. A.

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

van der Voort, H. T. M.

van Werkhoven, T.

Vazguez, J.

H. D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, “A human cell line from a pleural effusion derived from a breast carcinoma.” J. Natl. Cancer Inst. 51, 1409–1416 (1973).
[PubMed]

Verhaegen, M.

Visscher, K.

G. J. Brakenhoff, K. Visscher, H. T. M. Voort, Size and Shape of The Confocal Spot: Control and Relation to 3D Imaging and Image Processing, in Handbook of Biological Confocal Microscopy” J. B. Pawley, ed. (Plenum Press, New York, USA, 1990), pp. 87–91, revised ed.
[CrossRef]

von Zernike, F.

F. von Zernike, “Beugungstheorie des schneidenver-fahrens und seiner verbesserten form, der phasenkontrastmethode,” Physica 1, 689–704 (1934).
[CrossRef]

Voort, H. T. M.

G. J. Brakenhoff, K. Visscher, H. T. M. Voort, Size and Shape of The Confocal Spot: Control and Relation to 3D Imaging and Image Processing, in Handbook of Biological Confocal Microscopy” J. B. Pawley, ed. (Plenum Press, New York, USA, 1990), pp. 87–91, revised ed.
[CrossRef]

Vroom, J. M.

Wang, J.

Watanabe, T.

Webb, W. W.

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nature Biotechnol. 21, 1369–1377 (2003).
[CrossRef]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

Williams, D. R.

Williams, R. M.

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nature Biotechnol. 21, 1369–1377 (2003).
[CrossRef]

Wilson, T.

D. Débarre, E. J. Botcherby, T. Watanabe, S. Srinivas, M. J. Booth, T. Wilson, “Image-based adaptive optics for two-photon microscopy,” Opt. Lett. 34, 2495–2497 (2009).
[CrossRef] [PubMed]

D. Débarre, M. J. Booth, T. Wilson, “Image based adaptive optics through optimisation of low spatial frequencies,” Opt. Express 15, 8176–8190 (2007).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, T. Wilson, “Specimen-induced distortions in light microscopy,” J. Microsc. 228, 97–102 (2007).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, M. A. A. Neil, T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213, 11–19 (2004).
[CrossRef]

Wopat, L. M.

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

Zeng, J.

J. Zeng, P. Mahou, M.-C. Schanne-Klein, E. Beaurepaire, D. Débarre, “3D resolved mapping of optical aberrations in thick tissues,” Biomedical Opt. Express 3, 1898–1913 (2012).
[CrossRef]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nature Biotechnol. 21, 1369–1377 (2003).
[CrossRef]

Zuo, Y.

Annual Review of Biomedical Engineering (1)

P. T. C. So, C. Y. Dong, B. R. Masters, K. M. Berland, “Two-Photon Excitation Fluorescence Microscopy,” Annual Review of Biomedical Engineering 2, 399–429 (2000).
[CrossRef]

Appl. Opt. (5)

Astron. Astrophys. (1)

R. Foy, A. Labeyrie, “Feasibility of adaptive telescope with laser probe,” Astron. Astrophys. 152, L29–L31 (1985).

Biomed. Opt. Express (1)

Biomedical Opt. Express (1)

J. Zeng, P. Mahou, M.-C. Schanne-Klein, E. Beaurepaire, D. Débarre, “3D resolved mapping of optical aberrations in thick tissues,” Biomedical Opt. Express 3, 1898–1913 (2012).
[CrossRef]

Industrial Metrology (1)

M. Takeda, “Spatial-carrier fringe-pattern analysis and its applications to precision interferometry and profilometry: An overview,” Industrial Metrology 1, 79–99 (1990).
[CrossRef]

J. Biomed. Opt. (1)

J. W. Cha, J. Ballesta, P. T. C. So, “Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy,” J. Biomed. Opt. 15, 046022, 2010).
[CrossRef] [PubMed]

J. Cell Sci. (1)

D. M. Shotton, “Confocal scanning optical microscopy and its applications for biological specimens,” J. Cell Sci. 94, 175–206 (1989).

J. Microsc. (2)

M. Schwertner, M. J. Booth, M. A. A. Neil, T. Wilson, “Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry,” J. Microsc. 213, 11–19 (2004).
[CrossRef]

M. Schwertner, M. J. Booth, T. Wilson, “Specimen-induced distortions in light microscopy,” J. Microsc. 228, 97–102 (2007).
[CrossRef] [PubMed]

J. Natl. Cancer Inst. (1)

H. D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, “A human cell line from a pleural effusion derived from a breast carcinoma.” J. Natl. Cancer Inst. 51, 1409–1416 (1973).
[PubMed]

J. Opt. Soc. Am. (4)

J. Opt. Soc. Am. A (4)

Nature (1)

R. Q. Fugate, D. L. Fried, G. A. Ameer, B. R. Boeke, S. L. Browne, P. H. Roberts, R. E. Ruane, G. A. Tyler, L. M. Wopat, “Measurement of atmospheric wavefront distortion using scattered light from a laser guide-star,” Nature 353, 144–146 (1991).
[CrossRef]

Nature Biotechnol. (1)

W. R. Zipfel, R. M. Williams, W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nature Biotechnol. 21, 1369–1377 (2003).
[CrossRef]

Nature Protocols (1)

J. Friedrich, C. Seidel, R. Ebner, L. A. Kunz-Schughart, “Spheroid-based drug screen: considerations and practical approach,” Nature Protocols 4, 309–324 (2009).
[CrossRef] [PubMed]

Opt. Express (8)

R. Fiolka, K. Si, M. Cui, “Complex wavefront corrections for deep tissue focusing using low coherence backscattered light,” Opt. Express 20, 16532–16543 (2012).
[CrossRef]

S. Tuohy, A. G. Podoleanu, “Depth-resolved wavefront aberrations using a coherence-gated Shack-Hartmann wavefront sensor,” Opt. Express 18, 3458–3476 (2010).
[CrossRef] [PubMed]

P. Marsh, D. Burns, J. Girkin, “Practical implementation of adaptive optics in multiphoton microscopy,” Opt. Express 11, 1123–1130 (2003).
[CrossRef] [PubMed]

D. Débarre, M. J. Booth, T. Wilson, “Image based adaptive optics through optimisation of low spatial frequencies,” Opt. Express 15, 8176–8190 (2007).
[CrossRef] [PubMed]

A. Facomprez, E. Beaurepaire, D. Débarre, “Accuracy of correction in modal sensorless adaptive optics,” Opt. Express 20, 2598–2612 (2012).
[CrossRef] [PubMed]

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

X. Tao, J. Crest, S. Kotadia, O. Azucena, D. C. Chen, W. Sullivan, J. Kubby, “Live imaging using adaptive optics with fluorescent protein guide-stars,” Opt. Express 20, 15969, 2012).
[CrossRef] [PubMed]

C. Paterson, I. Munro, J. C. Dainty, “A low cost adaptive optics system using a membrane mirror,” Opt. Express 6, 175, 2000).
[CrossRef] [PubMed]

Opt. Lett. (7)

Philos. Trans. R. Soc., A (1)

M. J. Booth, “Adaptive optics in microscopy,” Philos. Trans. R. Soc., A 365, 2829–2843 (2007).
[CrossRef]

Physica (1)

F. von Zernike, “Beugungstheorie des schneidenver-fahrens und seiner verbesserten form, der phasenkontrastmethode,” Physica 1, 689–704 (1934).
[CrossRef]

Proc. Nat. Acad. Sci. USA (1)

M. Rückel, J. A. Mack-Bucher, W. Denk, “Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing,” Proc. Nat. Acad. Sci. USA 103, 17137–17142 (2006).
[CrossRef]

Science (2)

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, A. Et, “Optical coherence tomography,” Science 254, 1178–1181 (1991).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73–76 (1990).
[CrossRef] [PubMed]

The Computer Journal (1)

J. A. Nelder, R. Mead, “A Simplex Method for Function Minimization,” The Computer Journal 7, 308–313 (1965).
[CrossRef]

Other (6)

D. C. Ghiglia, M. D. Pritt, Two-Dimensional Phase Unwrapping: Theory, Algorithms, and Software, 1st ed. (Wiley-Interscience, 1998).

M. Peck, Interferometry mathematics, algorithms and data(2010).

M. Minsky, “Microscopy apparatus,” US Patent 3,013,467 (1961).

J. B. Pawley, ed., Handbook Of Biological Confocal Microscopy, 3rd ed. (SpringerUS, Boston, MA, 2006).
[CrossRef]

M. Feierabend, “Coherence-Gated Wave-Front Sensing in Strongly Scattering Samples,” Ph.D. thesis, Ruperto-Carola University of Heidelberg, Heidelberg, Germany (2004).

G. J. Brakenhoff, K. Visscher, H. T. M. Voort, Size and Shape of The Confocal Spot: Control and Relation to 3D Imaging and Image Processing, in Handbook of Biological Confocal Microscopy” J. B. Pawley, ed. (Plenum Press, New York, USA, 1990), pp. 87–91, revised ed.
[CrossRef]

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Figures (13)

Fig. 1
Fig. 1

Left: without sample, a plane wave focusses into a diffraction limited spot. Centre: in the presence of a perturbing sample, the focus is distorted leading to loss of signal and resolution. Right: using adaptive optics to pre-aberrate the wavefront, the focus can be improved.

Fig. 2
Fig. 2

Schematic optical setup showing the coherence-gated wavefront sensing principle. The pulsed laser beam is expanded (L1, L2) and then split (BS1) into a reference (long dashed) and a sample (solid) beam. In the sample beam the deformable mirror (DM) is re-imaged onto the objective’s back aperture (L3, L4). The sample is mounted on an XYZ piezo stage, and the fluorescence signal is separated by a dichroic beam splitter (DBS) and detected by a photomultiplier tube. The back-reflected light in the sample beam is again reflected by the DM and directed into the wavefront sensor (WFS) by BS1, where the back aperture is re-imaged onto the CCD (L6, L7), after being spatially filtered by the pinhole (PH). The reference beam’s optical path length and intensity are matched to that of the sample beam. The beams are combined on the CCD (BS2) where the temporally overlapping laser pulses interfere. By tilting folding mirrors in the reference beam (M6, M7) we tune the carrier frequency of the fringe pattern. Mirrors M3 and M4 are optional and are used to measure the influence of the DM for calibration purposes.

Fig. 3
Fig. 3

Excitation light entering from above is split into a reference and sample beam. In the sample, the light is reflected at different depths, but only back-scattered light from a thin layer, the coherence gate (solid red), has the same optical path length as the reference beam and will interfere to form fringes on the CCD. Pulses that originate from deeper or shallower in the sample (dashed red lines) arrive later or earlier (dashed red waves) than the reference pulse and do not contribute to the fringe pattern and add a static background.

Fig. 4
Fig. 4

Schematic illustration of the fringe analysis method. The fringe pattern recorded with the camera (top left) is Fourier transformed (top center). One sideband at f⃗0 is isolated and inverse transformed, yielding the complex wave (top right). The wave is propagated through a virtual Shack Hartman wavefront sensor to obtain a spot pattern (bottom right), which is reduced to a vector of spot displacements (bottom center). By multiplying this vector with a response matrix of a certain basis (e.g. Zernike), we obtain a modal description of the wavefront (bottom right).

Fig. 5
Fig. 5

Fringe and speckle patterns recorded from back-scattered light off the scattering beads solution (top) and off the spheroid sample (bottom). These images were observed by the WFS camera at the back aperture plane. The left images was obtained without a confocal pinhole, while the center and right images were taken with one in place. Additionally, the right images were flatfield corrected and interfered with the reference beam to produce fringes. The scattering bead solution shows a strong and homogeneous speckle pattern, while the spheroid shows larger speckles, indicating it is less scattering. The speckle pattern is spatially filtered by the confocal pinhole such that the fringe pattern contrast is enhanced, although larger-scale amplitude variations were still present. As can be seen in the center and right columns, the spatial frequency of the filtered speckles is similar for both samples. The fringe pattern has a higher spatial frequency than imposed by the confocal pinhole because the reference beam is introduced after the pinhole. All images were taken with the 20× objective and all fields of view are the same.

Fig. 6
Fig. 6

Correction of DM-induced aberrations Z5, Z6, Z11 (top) and Z5–Z8 (bottom) with the 20× objective in rat tail collagen fibres using the 20× objective. The panels show an image with the DM in aberrated shape (left), the system-corrected image (centre) and an image after aberration correction (right). For both cases, modes Z5–Z15 were controlled with the DM when correcting, and the sample was scanned over the area indicated by the dashed box. After correction we obtain images similar to the system-corrected DM case, although the even-only aberration correction (top) performed better in this case. The grayscale is identical in each row. The cross-section profile is taken along the arrow: dashed for aberrated, gray for system-corrected, and black for aberration-corrected data.

Fig. 7
Fig. 7

Correction loop diagnostics associated with Fig. 6: the top and bottom rows correspond to the respective rows in Fig. 6. Left: modal decomposition of deformable mirror shape during the correction experiment. The triangles note the initial DM shape and the squares indicate the final shape with respect to a system-corrected DM, the dots in between denote intermediate corrections. Right: norm of the DM shape during the experiment. The decay in the norm of the DM shape indicates that the correction is successful in recovering the system-corrected setting. See text for details.

Fig. 8
Fig. 8

DM-induced aberrations Z5–Z8 correction in a Hoechst-stained MCF7-spheroid sample using the 20× objective. The averaging area for the correction is indicated by the dashed box. Note that there is no observable bleaching in this area. The grayscale is identical, the cross-section profile is taken along the arrow.

Fig. 9
Fig. 9

Correction loop diagnostics associated with Fig. 8, plots as in Fig. 7. Left: modal decomposition of the DM shape during the experiment. Right: norm of the DM shape during the experiment.

Fig. 10
Fig. 10

Correction of DM-induced aberrations Z5, Z6, Z11 (top) and Z5–Z8 (bottom) in a Hoechst-stained MCF7-spheroid sample using the 40× objective. The corrected image (right) is shown with an aberrated (left) and a system-corrected reference (center) image. During correcting, the sample was scanned over the region indicated by the dashed box. The grayscale is identical in each row. The cross-section profile is taken along the arrow: dashed for aberrated, gray for system-corrected, and black for aberration-corrected data.

Fig. 11
Fig. 11

Correction loop diagnostics associated with Fig. 10, plots as in Fig. 7. Left: modal decomposition of the DM shape during the experiment. Right: norm of the DM shape during the experiment. See text for details.

Fig. 12
Fig. 12

Correction of sample-induced depth-dependent aberrations at 35 μm inside the spheroid imaged with the 40× objective. The bottom panels show a zoomed-in region of the top images, and were taken with the same corrected DM settings. Left is the flattened DM setting, right shows the image after correction. The dashed box indicates both the region shown in the bottom panels, as well as the area used to scan over during correction for averaging. The grayscale is identical per row. The cross-section profile is taken along the arrow: dashed indicates the system-corrected data, black the aberration-corrected data.

Fig. 13
Fig. 13

Correction loop diagnostics associated with Fig. 12, plots as in Fig. 7. Left: modal decomposition of the DM shape during the experiment. Right: norm of the DM shape during the experiment.

Equations (3)

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

Δ z = 2 d ph / M tan α
g ( x , y ) = a ( x , y ) + b ( x , y ) cos [ 2 π f 0 x + ϕ ( x , y ) ] ,
r = 1.22 λ / 2 n sin θ ,

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