Abstract

Optical aberrations deteriorate the performance of microscopes. Adaptive optics can be used to improve imaging performance via wavefront shaping. Here, we demonstrate a pupil-segmentation based adaptive optical approach with full-pupil illumination. When implemented in a two-photon fluorescence microscope, it recovers diffraction-limited performance and improves imaging signal and resolution.

© 2011 Optical Society of America

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References

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  1. M. J. Booth, Phil. Trans. R. Soc. A 365, 2829 (2007).
    [CrossRef] [PubMed]
  2. R. K. Tyson, Principles of Adaptive Optics (Academic, 1991).
  3. J. W. Hardy, Adaptive Optics for Astronomical Telescopes (Oxford University Press, 1998).
  4. N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141(2010).
    [CrossRef]
  5. N. Ji, T. R. Sato, and E. Betzig, “Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex,” Proc. Acad. Natl. Sci. USA , submitted for publication.
  6. S. I. Panagopoulou and D. R. Neal, J. Refract. Surg. 21, S563 (2005).
    [PubMed]
  7. M. A. Helmbrecht, M. He, C. J. Kempf, and M. Besse, Proc. SPIE 7931, 793108 (2011).
    [CrossRef]

2011 (1)

M. A. Helmbrecht, M. He, C. J. Kempf, and M. Besse, Proc. SPIE 7931, 793108 (2011).
[CrossRef]

2010 (1)

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141(2010).
[CrossRef]

2007 (1)

M. J. Booth, Phil. Trans. R. Soc. A 365, 2829 (2007).
[CrossRef] [PubMed]

2005 (1)

S. I. Panagopoulou and D. R. Neal, J. Refract. Surg. 21, S563 (2005).
[PubMed]

Besse, M.

M. A. Helmbrecht, M. He, C. J. Kempf, and M. Besse, Proc. SPIE 7931, 793108 (2011).
[CrossRef]

Betzig, E.

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141(2010).
[CrossRef]

N. Ji, T. R. Sato, and E. Betzig, “Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex,” Proc. Acad. Natl. Sci. USA , submitted for publication.

Booth, M. J.

M. J. Booth, Phil. Trans. R. Soc. A 365, 2829 (2007).
[CrossRef] [PubMed]

Hardy, J. W.

J. W. Hardy, Adaptive Optics for Astronomical Telescopes (Oxford University Press, 1998).

He, M.

M. A. Helmbrecht, M. He, C. J. Kempf, and M. Besse, Proc. SPIE 7931, 793108 (2011).
[CrossRef]

Helmbrecht, M. A.

M. A. Helmbrecht, M. He, C. J. Kempf, and M. Besse, Proc. SPIE 7931, 793108 (2011).
[CrossRef]

Ji, N.

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141(2010).
[CrossRef]

N. Ji, T. R. Sato, and E. Betzig, “Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex,” Proc. Acad. Natl. Sci. USA , submitted for publication.

Kempf, C. J.

M. A. Helmbrecht, M. He, C. J. Kempf, and M. Besse, Proc. SPIE 7931, 793108 (2011).
[CrossRef]

Milkie, D. E.

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141(2010).
[CrossRef]

Neal, D. R.

S. I. Panagopoulou and D. R. Neal, J. Refract. Surg. 21, S563 (2005).
[PubMed]

Panagopoulou, S. I.

S. I. Panagopoulou and D. R. Neal, J. Refract. Surg. 21, S563 (2005).
[PubMed]

Sato, T. R.

N. Ji, T. R. Sato, and E. Betzig, “Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex,” Proc. Acad. Natl. Sci. USA , submitted for publication.

Tyson, R. K.

R. K. Tyson, Principles of Adaptive Optics (Academic, 1991).

J. Refract. Surg. (1)

S. I. Panagopoulou and D. R. Neal, J. Refract. Surg. 21, S563 (2005).
[PubMed]

Nat. Methods (1)

N. Ji, D. E. Milkie, and E. Betzig, Nat. Methods 7, 141(2010).
[CrossRef]

Phil. Trans. R. Soc. A (1)

M. J. Booth, Phil. Trans. R. Soc. A 365, 2829 (2007).
[CrossRef] [PubMed]

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

N. Ji, T. R. Sato, and E. Betzig, “Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex,” Proc. Acad. Natl. Sci. USA , submitted for publication.

Proc. SPIE (1)

M. A. Helmbrecht, M. He, C. J. Kempf, and M. Besse, Proc. SPIE 7931, 793108 (2011).
[CrossRef]

Other (2)

R. K. Tyson, Principles of Adaptive Optics (Academic, 1991).

J. W. Hardy, Adaptive Optics for Astronomical Telescopes (Oxford University Press, 1998).

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Figures (4)

Fig. 1
Fig. 1

Schematic depicting the pupil-segmentation method with full-pupil illumination. (a) An ideal focus has all rays (blue and red) intersect at the same point, (b) whereas for an aberrated focus, rays do not. Scanning one of the rays (e.g., red ray) through a range of angles (shaded red cone) varies the intensity of the fluorescence excited at the focus. (c) Plotted as an image, this data exhibits an intensity extremum that is centered (dashed circle) over the scan region, provided the ray already intersects the ideal focus. (d) Shift of the extremum from the center indicates local wavefront tilt (dashed red line in b), which can be corrected by tilting the ray to the angle indicated by the extremum (solid red line in b).

Fig. 2
Fig. 2

(a) For an aberration-free system under full-pupil illumination, the signal maximum for each pupil segment occurs at the center of its own scanned region. (b) Aberration causes the signal extremum for each segment to shift away from this center. Segments marked with red or blue dots were taken with π / 2 or π initial phase offsets, respectively. (c) Images measured with single-segment illumination. (d) Axial images of a 2 μm diameter fluorescent bead without AO correction and with pupil-segmentation based AO under full-pupil and single- segment illumination, respectively. (e) The signal profile along the dotted line in (d). (f) The final corrective wavefront on the SLM obtained with full-pupil illumination, in wavelengths. Scale bar: 2 μm .

Fig. 3
Fig. 3

(a) Lateral and axial images of a 2 μm diameter bead without AO correction, with one, two, three rounds full-pupil illumination AO correction, and with single-segment illumination AO correction, respectively. The images without any AO correction (leftmost panel) have their intensity digitally enhanced 7.5 × to aid visualization. (b) The maximal signal increases with iterative full-pupil illumination AO correction. (c) The corrective wavefront on the SLM, in wavelengths. Scale bar: 2 μm .

Fig. 4
Fig. 4

(a) Maximal intensity projection in 30 μm depth of a dense fluorescent bead sample. (b) Axial images of a 2 μm diameter bead without AO, with full-pupil illumination, and with single-segment illumination AO. (c) Signal profile along the dotted line in (b). (d) Signal modulation during full-pupil illumination AO. (e) Images measured with single- pupil illumination AO. (f) Aberration in unit of wavelength. Scale bar: 2 μm .

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