Abstract

We describe a method of optical refocusing for high numerical aperture (NA) systems that is particularly relevant for confocal and multiphoton microscopy. This method avoids the spherical aberration that is common to other optical refocusing systems. We show that aberration-free images can be obtained over an axial scan range of 70μm for a 1.4 NA objective lens. As refocusing is implemented remotely from the specimen, this method will enable high axial scan speeds without mechanical interference between the objective lens and the specimen.

© 2007 Optical Society of America

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References

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  1. W. Göbel, B. M. Kampa, and F. Helmchen, Learn. Memory 4, 73 (2007).
  2. N. Callamaras and I. Parker, Cell Calcium 26, 271279 (1999).
    [CrossRef]
  3. M. Born and E. WolfPrinciples of Optics, 6th ed. (Pergamon, 1983).
  4. C. J. R. Sheppard and M. Gu, Appl. Opt. 30, 3563 (1991).
    [CrossRef] [PubMed]
  5. M. Petran, M. Hadravsky, M. D. Egger, and R. Galambos, J. Opt. Soc. Am. 58, 661 (1968).
    [CrossRef]

2007 (1)

W. Göbel, B. M. Kampa, and F. Helmchen, Learn. Memory 4, 73 (2007).

1999 (1)

N. Callamaras and I. Parker, Cell Calcium 26, 271279 (1999).
[CrossRef]

1991 (1)

1968 (1)

Born, M.

M. Born and E. WolfPrinciples of Optics, 6th ed. (Pergamon, 1983).

Callamaras, N.

N. Callamaras and I. Parker, Cell Calcium 26, 271279 (1999).
[CrossRef]

Egger, M. D.

Galambos, R.

Göbel, W.

W. Göbel, B. M. Kampa, and F. Helmchen, Learn. Memory 4, 73 (2007).

Gu, M.

Hadravsky, M.

Helmchen, F.

W. Göbel, B. M. Kampa, and F. Helmchen, Learn. Memory 4, 73 (2007).

Kampa, B. M.

W. Göbel, B. M. Kampa, and F. Helmchen, Learn. Memory 4, 73 (2007).

Parker, I.

N. Callamaras and I. Parker, Cell Calcium 26, 271279 (1999).
[CrossRef]

Petran, M.

Sheppard, C. J. R.

Wolf, E.

M. Born and E. WolfPrinciples of Optics, 6th ed. (Pergamon, 1983).

Appl. Opt. (1)

Cell Calcium (1)

N. Callamaras and I. Parker, Cell Calcium 26, 271279 (1999).
[CrossRef]

J. Opt. Soc. Am. (1)

Learn. Memory (1)

W. Göbel, B. M. Kampa, and F. Helmchen, Learn. Memory 4, 73 (2007).

Other (1)

M. Born and E. WolfPrinciples of Optics, 6th ed. (Pergamon, 1983).

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Figures (3)

Fig. 1
Fig. 1

Optical configurations for three defocusing systems: L1, L2, and L3, high NA objective lenses; M, planar mirror.

Fig. 2
Fig. 2

(a) x z sections of PSFs measured at focal positions of 0 and 12.5 μ m in the specimen. (b) x z sections of PSFs measured with the system in Fig. 1c at focal positions of 0 and 35.6 μ m in the specimen. All images are normalized to the maximum pixel value.

Fig. 3
Fig. 3

Fluorescence confocal x z sections of a mouse kidney specimen acquired using a tandem scanning system. (a) Refocusing was performed using the low NA system, equivalent to shifting the scanning unit. (b) Refocusing was performed by shifting the reference mirror in the focus of a high NA objective.

Equations (4)

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ϕ = n 1 k z cos θ = n 1 k z 1 s 2 ρ 2 ,
= n 1 k z ( 1 s 2 ρ 2 2 s 4 ρ 4 8 + ) ,
ϕ ( ρ ) = k Z C N A 1 2 2 M 1 2 ρ 2 ,
M = f 2 f 1 = n 2 F 2 M 1 n 1 F 1 M 2 ,

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