Abstract

Optical aberrations due to the inhomogeneous refractive index of tissue degrade the resolution and brightness of images in deep-tissue imaging. We introduce a confocal fluorescence microscope with adaptive optics, which can correct aberrations based on direct wavefront measurements using a Shack–Hartmann wavefront sensor with a fluorescent bead used as a point source reference beacon. The results show a 4.3× improvement in the Strehl ratio and a 240% improvement in the signal intensity for fixed mouse tissues at depths of up to 100μm.

© 2011 Optical Society of America

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References

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2010

2007

2006

M. Rueckel, J. A. Mack-Bucher, and W. Denk, Proc. Natl. Acad. Sci. USA 103, 17137 (2006).
[CrossRef] [PubMed]

2004

M. Reicherter, W. Gorski, T. Haist, and W. Osten, Proc. SPIE 5462, 68 (2004).
[CrossRef]

2003

2002

J. L. Beverage, R. V. Shack, and M. R. Descour, J. Microsc. 205, 61 (2002).
[CrossRef] [PubMed]

1999

1994

P. Menei, A. Croue, V. Daniel, A. Pouplard-Barthelaix, and J. P. Benoit, J. Biomed. Mater. Res. 28, 1079 (1994).
[CrossRef] [PubMed]

1953

H. W. Babcock, Publ. Astron. Soc. Pac. 65, 229 (1953).
[CrossRef]

Awwal, A. A. S.

J. Porter, H. Queener, J. Lin, K. Thorn, and A. A. S. Awwal, Adaptive Optics for Vision Science: Principles, Practices, Design and Applications (Wiley, 2006).
[CrossRef]

Azucena, O.

Babcock, H. W.

H. W. Babcock, Publ. Astron. Soc. Pac. 65, 229 (1953).
[CrossRef]

Benoit, J. P.

P. Menei, A. Croue, V. Daniel, A. Pouplard-Barthelaix, and J. P. Benoit, J. Biomed. Mater. Res. 28, 1079 (1994).
[CrossRef] [PubMed]

Betzig, E.

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

Beverage, J. L.

J. L. Beverage, R. V. Shack, and M. R. Descour, J. Microsc. 205, 61 (2002).
[CrossRef] [PubMed]

Bifano, T. G.

Biss, D. P.

Booth, M. J.

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

Burns, D.

Burns, S. A.

Cao, J.

Côté, D.

Crest, J.

Croue, A.

P. Menei, A. Croue, V. Daniel, A. Pouplard-Barthelaix, and J. P. Benoit, J. Biomed. Mater. Res. 28, 1079 (1994).
[CrossRef] [PubMed]

Dainty, J. C.

Daniel, V.

P. Menei, A. Croue, V. Daniel, A. Pouplard-Barthelaix, and J. P. Benoit, J. Biomed. Mater. Res. 28, 1079 (1994).
[CrossRef] [PubMed]

Denk, W.

M. Rueckel, J. A. Mack-Bucher, and W. Denk, Proc. Natl. Acad. Sci. USA 103, 17137 (2006).
[CrossRef] [PubMed]

Descour, M. R.

J. L. Beverage, R. V. Shack, and M. R. Descour, J. Microsc. 205, 61 (2002).
[CrossRef] [PubMed]

Diaz, L.

Dillon, D.

Gavel, D.

Girkin, J.

Gorski, W.

M. Reicherter, W. Gorski, T. Haist, and W. Osten, Proc. SPIE 5462, 68 (2004).
[CrossRef]

Haist, T.

M. Reicherter, W. Gorski, T. Haist, and W. Osten, Proc. SPIE 5462, 68 (2004).
[CrossRef]

Haro, S.

Ji, N.

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

Kner, P.

Kubby, J.

Lin, C. P.

Lin, J.

J. Porter, H. Queener, J. Lin, K. Thorn, and A. A. S. Awwal, Adaptive Optics for Vision Science: Principles, Practices, Design and Applications (Wiley, 2006).
[CrossRef]

Mack-Bucher, J. A.

M. Rueckel, J. A. Mack-Bucher, and W. Denk, Proc. Natl. Acad. Sci. USA 103, 17137 (2006).
[CrossRef] [PubMed]

Marsh, P.

Menei, P.

P. Menei, A. Croue, V. Daniel, A. Pouplard-Barthelaix, and J. P. Benoit, J. Biomed. Mater. Res. 28, 1079 (1994).
[CrossRef] [PubMed]

Milkie, D. E.

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

Olivier, S.

Osten, W.

M. Reicherter, W. Gorski, T. Haist, and W. Osten, Proc. SPIE 5462, 68 (2004).
[CrossRef]

Pawley, J. B.

J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006).
[CrossRef]

Porter, J.

J. Porter, H. Queener, J. Lin, K. Thorn, and A. A. S. Awwal, Adaptive Optics for Vision Science: Principles, Practices, Design and Applications (Wiley, 2006).
[CrossRef]

Pouplard-Barthelaix, A.

P. Menei, A. Croue, V. Daniel, A. Pouplard-Barthelaix, and J. P. Benoit, J. Biomed. Mater. Res. 28, 1079 (1994).
[CrossRef] [PubMed]

Queener, H.

J. Porter, H. Queener, J. Lin, K. Thorn, and A. A. S. Awwal, Adaptive Optics for Vision Science: Principles, Practices, Design and Applications (Wiley, 2006).
[CrossRef]

Reicherter, M.

M. Reicherter, W. Gorski, T. Haist, and W. Osten, Proc. SPIE 5462, 68 (2004).
[CrossRef]

Rueckel, M.

M. Rueckel, J. A. Mack-Bucher, and W. Denk, Proc. Natl. Acad. Sci. USA 103, 17137 (2006).
[CrossRef] [PubMed]

Shack, R. V.

J. L. Beverage, R. V. Shack, and M. R. Descour, J. Microsc. 205, 61 (2002).
[CrossRef] [PubMed]

Sullivan, W.

Sumorok, D.

Thorn, K.

J. Porter, H. Queener, J. Lin, K. Thorn, and A. A. S. Awwal, Adaptive Optics for Vision Science: Principles, Practices, Design and Applications (Wiley, 2006).
[CrossRef]

Tyson, R. K.

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

Veilleux, I.

Webb, R. H.

Zamiri, P.

Zhou, Y.

J. Biomed. Mater. Res.

P. Menei, A. Croue, V. Daniel, A. Pouplard-Barthelaix, and J. P. Benoit, J. Biomed. Mater. Res. 28, 1079 (1994).
[CrossRef] [PubMed]

J. Microsc.

J. L. Beverage, R. V. Shack, and M. R. Descour, J. Microsc. 205, 61 (2002).
[CrossRef] [PubMed]

Nat. Meth.

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

Opt. Express

Opt. Lett.

Phil. Trans. R. Soc. A

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

Proc. Natl. Acad. Sci. USA

M. Rueckel, J. A. Mack-Bucher, and W. Denk, Proc. Natl. Acad. Sci. USA 103, 17137 (2006).
[CrossRef] [PubMed]

Proc. SPIE

M. Reicherter, W. Gorski, T. Haist, and W. Osten, Proc. SPIE 5462, 68 (2004).
[CrossRef]

Publ. Astron. Soc. Pac.

H. W. Babcock, Publ. Astron. Soc. Pac. 65, 229 (1953).
[CrossRef]

Other

J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 2006).
[CrossRef]

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

J. Porter, H. Queener, J. Lin, K. Thorn, and A. A. S. Awwal, Adaptive Optics for Vision Science: Principles, Practices, Design and Applications (Wiley, 2006).
[CrossRef]

Supplementary Material (2)

» Media 1: AVI (1099 KB)     
» Media 2: AVI (1800 KB)     

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Figures (4)

Fig. 1
Fig. 1

AO confocal microscope: an He–Ne laser emits light at 633 nm for excitation of the fluorescent reference beacons. Light emitted from the reference beacon is passed through filter F1 to the wavefront sensor. A solid-state laser emits light at 515 nm that excites the YFP bred into the sample. The emission light is filtered by F3 and detected by the photomultiplier tube (PMT). The wavefront aberrations are corrected by the DM.

Fig. 2
Fig. 2

Wavefront measurements from a fluorescent microsphere. Wavefront error (a) before and (b) after correction. The rms errors for (a) and (b) are 0.24 λ and 0.028 λ ( λ = 633 nm ), respectively. Images of the microsphere (c) before and (d) after correction.

Fig. 3
Fig. 3

Confocal fluorescence imaging through 100 μm thick mouse brain tissue. The maximum intensity projection image from the bottom of the tissue (a) before and (b) after correction. The dashed boxes indicate the enlarged image (c) before and (d) after correction. (e) Intensity profiles along the dashed curve in the uncorrected image (c) and along the solid curve in the corrected image (d). The scale bar is 5 μm .

Fig. 4
Fig. 4

Enlarged 3D volume images of solid box in Fig. 3 (a) before (Media 1) and (b) after correction (Media 2).

Tables (1)

Tables Icon

Table 1 Statistical Properties of the Strehl Ratio for Brain Tissues

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