Abstract

The scanning two-photon fluorescence microscope produces optically sectioned images from the focal plane. It is sometimes desirable to acquire images from other planes of the specimen that are inclined with respect to the focal plane. In this Letter, we discuss the issues concerned with acquiring such images together with the effects of the inclination angle on image resolution and sectioning strength. To obtain images from oblique planes at high speed, a two-photon system was built wherein a novel optical system is used to provide aberration-free scanning.

© 2011 Optical Society of America

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References

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  1. G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
    [CrossRef] [PubMed]
  2. W. Göbel and F. Helmchen, J. Neurophysiol. 98, 3770(2007).
    [CrossRef] [PubMed]
  3. E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Lett. 32, 2007 (2007).
    [CrossRef] [PubMed]
  4. E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Commun. 281, 880 (2008).
    [CrossRef]
  5. M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, 1996).
    [CrossRef]
  6. J. W. Goodman, Introduction to Fourier Optics, 2nd ed. (McGraw-Hill, 1996).
  7. E. Botcherby, C. Smith, M. Booth, R. Juškaitis, and T. Wilson, Proc. SPIE 7569, 756917 (2010).
    [CrossRef]

2010

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

E. Botcherby, C. Smith, M. Booth, R. Juškaitis, and T. Wilson, Proc. SPIE 7569, 756917 (2010).
[CrossRef]

2008

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Commun. 281, 880 (2008).
[CrossRef]

2007

Bollensdorff, C.

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

Booth, M.

E. Botcherby, C. Smith, M. Booth, R. Juškaitis, and T. Wilson, Proc. SPIE 7569, 756917 (2010).
[CrossRef]

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Commun. 281, 880 (2008).
[CrossRef]

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Lett. 32, 2007 (2007).
[CrossRef] [PubMed]

Botcherby, E.

E. Botcherby, C. Smith, M. Booth, R. Juškaitis, and T. Wilson, Proc. SPIE 7569, 756917 (2010).
[CrossRef]

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Commun. 281, 880 (2008).
[CrossRef]

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Lett. 32, 2007 (2007).
[CrossRef] [PubMed]

Bub, G.

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

Burton, R.

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

Camelliti, P.

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

Clarke, K.

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

Göbel, W.

W. Göbel and F. Helmchen, J. Neurophysiol. 98, 3770(2007).
[CrossRef] [PubMed]

Goodman, J. W.

J. W. Goodman, Introduction to Fourier Optics, 2nd ed. (McGraw-Hill, 1996).

Gu, M.

M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, 1996).
[CrossRef]

Helmchen, F.

W. Göbel and F. Helmchen, J. Neurophysiol. 98, 3770(2007).
[CrossRef] [PubMed]

Juškaitis, R.

E. Botcherby, C. Smith, M. Booth, R. Juškaitis, and T. Wilson, Proc. SPIE 7569, 756917 (2010).
[CrossRef]

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Commun. 281, 880 (2008).
[CrossRef]

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Lett. 32, 2007 (2007).
[CrossRef] [PubMed]

Kohl, P.

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

Picton, G.

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

Smith, C.

E. Botcherby, C. Smith, M. Booth, R. Juškaitis, and T. Wilson, Proc. SPIE 7569, 756917 (2010).
[CrossRef]

Stuckey, D.

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

Wilson, T.

E. Botcherby, C. Smith, M. Booth, R. Juškaitis, and T. Wilson, Proc. SPIE 7569, 756917 (2010).
[CrossRef]

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Commun. 281, 880 (2008).
[CrossRef]

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Lett. 32, 2007 (2007).
[CrossRef] [PubMed]

Am. J. Physiol. Heart. Circ. Physiol.

G. Bub, P. Camelliti, C. Bollensdorff, D. Stuckey, G. Picton, R. Burton, K. Clarke, and P. Kohl, Am. J. Physiol. Heart. Circ. Physiol. 298, H1616 (2010).
[CrossRef] [PubMed]

J. Neurophysiol.

W. Göbel and F. Helmchen, J. Neurophysiol. 98, 3770(2007).
[CrossRef] [PubMed]

Opt. Commun.

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, Opt. Commun. 281, 880 (2008).
[CrossRef]

Opt. Lett.

Proc. SPIE

E. Botcherby, C. Smith, M. Booth, R. Juškaitis, and T. Wilson, Proc. SPIE 7569, 756917 (2010).
[CrossRef]

Other

M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, 1996).
[CrossRef]

J. W. Goodman, Introduction to Fourier Optics, 2nd ed. (McGraw-Hill, 1996).

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Figures (3)

Fig. 1
Fig. 1

(a) Imaging oblique plane with angle θ. (b) 3D OTF of the system, rotationally symmetric r. (c) Spatial frequency content in oblique-plane image. (Projection of 3D OTF along the r axis.) (d) and (e) Spatial frequency cutoff along inclined axis and section thickness when imaging an oblique plane for 1.15 NA (solid curve) and 0.8 NA (broken curve) water immersion lenses. λ = 850 nm .

Fig. 2
Fig. 2

Oblique-plane scanning multiphoton microscope. The LSU scans the focal spot laterally in the specimen, and the ASU scans the spot axially. Oblique planes are scanned in three dimensions by using both the LSU and ASU.

Fig. 3
Fig. 3

Oblique-plane images of a pollen grain with θ = 0 ° , 45 ° , and 90 ° along with their corresponding 2D FFT plots. Calculated regions of support are marked in each case. 512 × 512 pixels, 10 μs per pixel.

Equations (2)

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| r | = { 1 , 0 l 2 , l 1 4 l 2 , 2 < l 4 ,
I ( z ) = | h ( x , y ) | 4 d x d y ,

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