Abstract

A dedicated two-photon microscope incorporating adaptive-optic correction of specimen-induced aberrations is presented. Wavefront alteration of the scanning laser beam was achieved by use of a micromachined deformable mirror. Post scan head implementation produces a compact module compatible with the Bio-Rad MRC-600 scan head. Automatic aberration correction using feedback from the multiphoton fluorescence intensity allowed the adaptive optic to extend the imaging depth attainable in both artificial and biological refractive-index mismatched samples. With a 1.3-NA, ×40, Nikon oil immersion objective, the imaging depth in water was extended from approximately 3.4 to 46.2 µm with a resolution defined by a FWHM axial point-spread function of 1.25 µm.

© 2003 Optical Society of America

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References

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  7. L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, �??Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror,�?? J. Microsc. 206, 65-71 (2002).
    [CrossRef] [PubMed]
  8. M. J. Booth, M. A. A. Neil, R. Juskaitis, and T. Wilson, �??Adaptive aberration correction in a confocal microscope,�?? PNAS 99, 9, 5788-5792 (2002).
    [CrossRef]
  9. G. Vdovin, S. Middelhoek, and P. Sarro, �??Technology and applications of micromachined silicon adaptive mirrors,�?? Opt. Eng. 36, 1382-1390 (1997).
    [CrossRef]

Appl. Opt.

J. Microsc.

D. S. Wan, M. Rajadhyaksha, and R. H. Webb, �??Analysis of spherical aberration of a water immersion objective: application to specimens with refractive index 1.33-1.40,�?? J. Microsc. 197, 274-284 (2000).
[CrossRef] [PubMed]

M. J. Booth and T. Wilson, �??Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin,�?? J. Microsc. 200, 68-74 (2000).
[CrossRef] [PubMed]

M. A. A. Neil, R. Juskaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata, �??Adaptive aberration correction in a two-photon microscope,�?? J. Microsc. 200, 105-108 (2000).
[CrossRef] [PubMed]

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris, �??Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror,�?? J. Microsc. 206, 65-71 (2002).
[CrossRef] [PubMed]

Opt. Eng.

G. Vdovin, S. Middelhoek, and P. Sarro, �??Technology and applications of micromachined silicon adaptive mirrors,�?? Opt. Eng. 36, 1382-1390 (1997).
[CrossRef]

Other

A. Diaspro, Confocal and Two-Photon Microscopy, Foundations, Applications and Advances (Wiley-Liss, New York, 2002).

M. J. Booth, M. A. A. Neil, R. Juskaitis, and T. Wilson, �??Adaptive aberration correction in a confocal microscope,�?? PNAS 99, 9, 5788-5792 (2002).
[CrossRef]

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

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Figures (6)

Fig. 1.
Fig. 1.

Experimental apparatus.

Fig. 2.
Fig. 2.

xy scan of 105nm bead before (a) and after correction (b). Image dimensions are 5.0 µm × 2.3 µm.

Fig. 3.
Fig. 3.

xz scan of a 105nm bead just under the coverslip (a) and a bead at a water depth of 25.7µm before (b) and after correction (c). Image widths are 3.8µm with the scan depths being 2.1, 4.3 and 2.4µm, respectively.

Fig. 4.
Fig. 4.

FWHM of the axial psf as a function of water depth with and without correction.

Fig. 5.
Fig. 5.

Feature in tissue imaged using mirror shape A (a) and mirror shape B (b). Image dimensions are 32 µm × 32 µm.

Fig. 6.
Fig. 6.

Signal vs. objective movement into smooth muscle cells.

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