Abstract

We describe an optical microscope system whose focal setting can be changed quickly without moving the objective lens or specimen. Using this system, diffraction limited images can be acquired from a wide range of focal settings without introducing optical aberrations that degrade image quality. We combine this system with a real time Nipkow disc based confocal microscope so as to permit the acquisition of extended depth of field images directly in a single frame of the CCD camera. We also demonstrate a simple modification that enables extended depth of field images to be acquired from different angles of perspective, where the angle can be changed over a continuous range by the user in real-time.

© 2008 Optical Society of America

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References

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  1. T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic Press, London, 1984).
  2. M. A. A. Neil, R. Ju¡skaitis, T. Wilson, Z. J. Laczik, and V. Sarafis, "Optimized pupil-plane filters for confocal microscope point-spread function engineering," Opt. Lett. 25,245-247 (2000).
    [CrossRef]
  3. E. Botcherby, R. Ju¡skaitis, and T. Wilson, "Scanning two photon fluorescence microscopy with extended depth of field," Opt. Comm. 268,253-260 (2006).
    [CrossRef]
  4. N. George and W. Chi, "Extended depth of field using a logarithmic asphere," J. Opt. A: Pure Appl. Opt. 5,S157-S163 (2003).
    [CrossRef]
  5. E. R. Dowski and W. T. Cathey, "Extended depth of field through wave-front coding," Appl. Opt. 34,1859-1866 (1995).
    [CrossRef] [PubMed]
  6. M. Born and E. Wolf, Principles of Optics (Pergamon Press, 6th edition, 1983).
  7. E. Botcherby, R. Ju¡skaitis, M. Booth, and T. Wilson, "Aberration-free optical refocusing in high numerical aperture microscopy," Opt. Lett. 32,2007-2009 (2007).
    [CrossRef] [PubMed]
  8. E. Botcherby, R. Ju¡skaitis, M. Booth, and T. Wilson, "An optical technique for remote focusing in microscopy," Opt. Comm. 281,880-887 (2008).
    [CrossRef]

2008

E. Botcherby, R. Ju¡skaitis, M. Booth, and T. Wilson, "An optical technique for remote focusing in microscopy," Opt. Comm. 281,880-887 (2008).
[CrossRef]

2007

2006

E. Botcherby, R. Ju¡skaitis, and T. Wilson, "Scanning two photon fluorescence microscopy with extended depth of field," Opt. Comm. 268,253-260 (2006).
[CrossRef]

2003

N. George and W. Chi, "Extended depth of field using a logarithmic asphere," J. Opt. A: Pure Appl. Opt. 5,S157-S163 (2003).
[CrossRef]

2000

1995

Botcherby, E.

E. Botcherby, R. Ju¡skaitis, M. Booth, and T. Wilson, "An optical technique for remote focusing in microscopy," Opt. Comm. 281,880-887 (2008).
[CrossRef]

E. Botcherby, R. Ju¡skaitis, M. Booth, and T. Wilson, "Aberration-free optical refocusing in high numerical aperture microscopy," Opt. Lett. 32,2007-2009 (2007).
[CrossRef] [PubMed]

E. Botcherby, R. Ju¡skaitis, and T. Wilson, "Scanning two photon fluorescence microscopy with extended depth of field," Opt. Comm. 268,253-260 (2006).
[CrossRef]

Cathey, W. T.

Chi, W.

N. George and W. Chi, "Extended depth of field using a logarithmic asphere," J. Opt. A: Pure Appl. Opt. 5,S157-S163 (2003).
[CrossRef]

Dowski, E. R.

George, N.

N. George and W. Chi, "Extended depth of field using a logarithmic asphere," J. Opt. A: Pure Appl. Opt. 5,S157-S163 (2003).
[CrossRef]

Neil, M. A. A.

Appl. Opt.

J. Opt. A: Pure Appl. Opt.

N. George and W. Chi, "Extended depth of field using a logarithmic asphere," J. Opt. A: Pure Appl. Opt. 5,S157-S163 (2003).
[CrossRef]

Opt. Comm.

E. Botcherby, R. Ju¡skaitis, and T. Wilson, "Scanning two photon fluorescence microscopy with extended depth of field," Opt. Comm. 268,253-260 (2006).
[CrossRef]

E. Botcherby, R. Ju¡skaitis, M. Booth, and T. Wilson, "An optical technique for remote focusing in microscopy," Opt. Comm. 281,880-887 (2008).
[CrossRef]

Opt. Lett.

Other

T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic Press, London, 1984).

M. Born and E. Wolf, Principles of Optics (Pergamon Press, 6th edition, 1983).

Supplementary Material (1)

» Media 1: MOV (136 KB)     

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Figures (4)

Fig. 1.
Fig. 1.

(a) A standard microscope system and (b) a new architecture that permits remote focusing. In this arrangement the image plane is conjugate to an effective focal plane, whose axial position ΔZ depends on the position of mirror M. See main text for the definition of abbreviations.

Fig. 2.
Fig. 2.

Fluorescence confocal imaging system using a Nipkow disc. See main text for the definition of abbreviations.

Fig. 3.
Fig. 3.

Focusing 15µm below the surface of a mouse kidney specimen by (a) focusing with the new architecture, (b) moving the specimen itself and (c) by adjusting the image conjugate.

Fig. 4.
Fig. 4.

(a) Trajectory of R during the acquisition of a single EDF image, (b) the trajectory of R when M2 is tilted in synchrony with the focusing action, (c) a single frame from an EDF movie of a pollen grain and (d) an EDF movie of a pollen grain where the perspective is changed by the user in real-time (Media 1).

Equations (2)

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Δ Z = 2 n 2 n 1 Δ z ,
Δ X = f tan ( Δ θ ) f Δ θ ,

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