Abstract

A confocal microscope that uses separate, noncollinear objective lenses for illumination and collection makes it possible to work 20 mm from a sample with a field of view of 2.25 mm across and still achieve lateral and axial resolutions below 5 µm. The design is expected to have application to in vivo confocal microscopy, allowing a field of view and section thickness similar to those used by pathologists in examining excised tissue.

© 1999 Optical Society of America

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References

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  1. J. Pawley, ed., Handbook of Biological Confocal Microscopy, 3rd ed. (Plenum, New York, 1996); T. Wilson, ed. Confocal Microscopy (Academic, London, 1990).
  2. T. J. Flotte, Massachusetts General Hospital, Boston, Mass. 02114 (personal communication, 1997–1999), confirmed W. Hammond (Denver, Colo., 1998).
  3. S. Lindek, E. H. K. Stelzer, “Optical transfer functions for confocal theta fluorescence microscopy,” J. Opt. Soc. Am. 13, 479–482 (1996); E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994); “A new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopy,” J. Microsc. 179, 1–10 (1995); S. W. Hell, M. Schrader, H. T. M. Van Der Voort, “Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range,” J. Microsc. 187, 1–7 (1997).
    [CrossRef] [PubMed]
  4. S. Inoue, K. R. Spring, Video Microscopy, The Fundamentals (Plenum, New York, 1997).
    [CrossRef]
  5. Ref. 1, pp. 1–3. See also Ref. 6.
  6. R. H. Webb, “Cofocal optical microscopy,” Rep. Prog. Phys. 59, 427–471 (1996).
    [CrossRef]
  7. R. H. Webb, “Optics for laser rasters,” Appl. Opt. 23, 3680–3683 (1984).
    [CrossRef] [PubMed]
  8. Y. Li, “Laser beam scanning by rotary mirrors. II. Conic section scan patterns,” Appl. Opt. 34, 6417–6430 (1995).
    [CrossRef] [PubMed]
  9. Geltech, Inc., 3267 Progress Dr., Orlando, Fla. 32826.
  10. N. Takeshita, T. Fujita, K. Kime, “Dynamic characteristics of lens actuator for digital video disc,” in Optical Data Storage, G. R. Knight, H. Ooki, S. Tyan, eds., Proc. SPIE2514, 159–166 (1995); Y. Kano, T. Kohki, T. Watanabe, “Characteristics of linear DC motor drive X-Y stage within position detector for microscope,” Trans. Inst. Electron. Commun. Eng. Jpn. Sect. D 112, 1220–1225 (1992).
  11. M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995).
    [CrossRef] [PubMed]
  12. W.-F. Cheong, S. A. Prahl, A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
    [CrossRef]

1996 (2)

S. Lindek, E. H. K. Stelzer, “Optical transfer functions for confocal theta fluorescence microscopy,” J. Opt. Soc. Am. 13, 479–482 (1996); E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994); “A new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopy,” J. Microsc. 179, 1–10 (1995); S. W. Hell, M. Schrader, H. T. M. Van Der Voort, “Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range,” J. Microsc. 187, 1–7 (1997).
[CrossRef] [PubMed]

R. H. Webb, “Cofocal optical microscopy,” Rep. Prog. Phys. 59, 427–471 (1996).
[CrossRef]

1995 (2)

Y. Li, “Laser beam scanning by rotary mirrors. II. Conic section scan patterns,” Appl. Opt. 34, 6417–6430 (1995).
[CrossRef] [PubMed]

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995).
[CrossRef] [PubMed]

1990 (1)

W.-F. Cheong, S. A. Prahl, A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

1984 (1)

Anderson, R. R.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995).
[CrossRef] [PubMed]

Cheong, W.-F.

W.-F. Cheong, S. A. Prahl, A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Esterowitz, D.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995).
[CrossRef] [PubMed]

Flotte, T. J.

T. J. Flotte, Massachusetts General Hospital, Boston, Mass. 02114 (personal communication, 1997–1999), confirmed W. Hammond (Denver, Colo., 1998).

Fujita, T.

N. Takeshita, T. Fujita, K. Kime, “Dynamic characteristics of lens actuator for digital video disc,” in Optical Data Storage, G. R. Knight, H. Ooki, S. Tyan, eds., Proc. SPIE2514, 159–166 (1995); Y. Kano, T. Kohki, T. Watanabe, “Characteristics of linear DC motor drive X-Y stage within position detector for microscope,” Trans. Inst. Electron. Commun. Eng. Jpn. Sect. D 112, 1220–1225 (1992).

Grossman, M.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995).
[CrossRef] [PubMed]

Inoue, S.

S. Inoue, K. R. Spring, Video Microscopy, The Fundamentals (Plenum, New York, 1997).
[CrossRef]

Kime, K.

N. Takeshita, T. Fujita, K. Kime, “Dynamic characteristics of lens actuator for digital video disc,” in Optical Data Storage, G. R. Knight, H. Ooki, S. Tyan, eds., Proc. SPIE2514, 159–166 (1995); Y. Kano, T. Kohki, T. Watanabe, “Characteristics of linear DC motor drive X-Y stage within position detector for microscope,” Trans. Inst. Electron. Commun. Eng. Jpn. Sect. D 112, 1220–1225 (1992).

Li, Y.

Lindek, S.

S. Lindek, E. H. K. Stelzer, “Optical transfer functions for confocal theta fluorescence microscopy,” J. Opt. Soc. Am. 13, 479–482 (1996); E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994); “A new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopy,” J. Microsc. 179, 1–10 (1995); S. W. Hell, M. Schrader, H. T. M. Van Der Voort, “Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range,” J. Microsc. 187, 1–7 (1997).
[CrossRef] [PubMed]

Prahl, S. A.

W.-F. Cheong, S. A. Prahl, A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Rajadhyaksha, M.

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995).
[CrossRef] [PubMed]

Spring, K. R.

S. Inoue, K. R. Spring, Video Microscopy, The Fundamentals (Plenum, New York, 1997).
[CrossRef]

Stelzer, E. H. K.

S. Lindek, E. H. K. Stelzer, “Optical transfer functions for confocal theta fluorescence microscopy,” J. Opt. Soc. Am. 13, 479–482 (1996); E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994); “A new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopy,” J. Microsc. 179, 1–10 (1995); S. W. Hell, M. Schrader, H. T. M. Van Der Voort, “Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range,” J. Microsc. 187, 1–7 (1997).
[CrossRef] [PubMed]

Takeshita, N.

N. Takeshita, T. Fujita, K. Kime, “Dynamic characteristics of lens actuator for digital video disc,” in Optical Data Storage, G. R. Knight, H. Ooki, S. Tyan, eds., Proc. SPIE2514, 159–166 (1995); Y. Kano, T. Kohki, T. Watanabe, “Characteristics of linear DC motor drive X-Y stage within position detector for microscope,” Trans. Inst. Electron. Commun. Eng. Jpn. Sect. D 112, 1220–1225 (1992).

Webb, R. H.

R. H. Webb, “Cofocal optical microscopy,” Rep. Prog. Phys. 59, 427–471 (1996).
[CrossRef]

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995).
[CrossRef] [PubMed]

R. H. Webb, “Optics for laser rasters,” Appl. Opt. 23, 3680–3683 (1984).
[CrossRef] [PubMed]

Welch, A. J.

W.-F. Cheong, S. A. Prahl, A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

Appl. Opt. (2)

IEEE J. Quantum Electron. (1)

W.-F. Cheong, S. A. Prahl, A. J. Welch, “A review of the optical properties of biological tissues,” IEEE J. Quantum Electron. 26, 2166–2185 (1990).
[CrossRef]

J. Invest. Dermatol. (1)

M. Rajadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, R. R. Anderson, “In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast,” J. Invest. Dermatol. 104, 946–952 (1995).
[CrossRef] [PubMed]

J. Opt. Soc. Am. (1)

S. Lindek, E. H. K. Stelzer, “Optical transfer functions for confocal theta fluorescence microscopy,” J. Opt. Soc. Am. 13, 479–482 (1996); E. H. K. Stelzer, S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111, 536–547 (1994); “A new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopy,” J. Microsc. 179, 1–10 (1995); S. W. Hell, M. Schrader, H. T. M. Van Der Voort, “Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range,” J. Microsc. 187, 1–7 (1997).
[CrossRef] [PubMed]

Rep. Prog. Phys. (1)

R. H. Webb, “Cofocal optical microscopy,” Rep. Prog. Phys. 59, 427–471 (1996).
[CrossRef]

Other (6)

S. Inoue, K. R. Spring, Video Microscopy, The Fundamentals (Plenum, New York, 1997).
[CrossRef]

Ref. 1, pp. 1–3. See also Ref. 6.

J. Pawley, ed., Handbook of Biological Confocal Microscopy, 3rd ed. (Plenum, New York, 1996); T. Wilson, ed. Confocal Microscopy (Academic, London, 1990).

T. J. Flotte, Massachusetts General Hospital, Boston, Mass. 02114 (personal communication, 1997–1999), confirmed W. Hammond (Denver, Colo., 1998).

Geltech, Inc., 3267 Progress Dr., Orlando, Fla. 32826.

N. Takeshita, T. Fujita, K. Kime, “Dynamic characteristics of lens actuator for digital video disc,” in Optical Data Storage, G. R. Knight, H. Ooki, S. Tyan, eds., Proc. SPIE2514, 159–166 (1995); Y. Kano, T. Kohki, T. Watanabe, “Characteristics of linear DC motor drive X-Y stage within position detector for microscope,” Trans. Inst. Electron. Commun. Eng. Jpn. Sect. D 112, 1220–1225 (1992).

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Figures (11)

Fig. 1
Fig. 1

Point-spread functions of low NA lenses concentrate energy laterally better than axially, but when angled, their overlap volume is more nearly the same in both directions. Contours are at 5% and 50% of peak intensity. The objectives are not to scale (they would be 20 mm away).

Fig. 2
Fig. 2

Point-spread functions of a single objective lens. The straight lines on the left are the extent of the geometric shadow (NA is 0.2). Next is the point-spread function of that lens. Multiplying the point-spread function by itself produces the confocal point-spread function shown on the right. Contours are at 5% and 50% of peak intensity.

Fig. 3
Fig. 3

Two objectives at 60 deg will view a point. That point can be made a line perpendicular to the paper by rotating a mirror in a pupillary conjugate. Scanning the other axes will eventually be done by moving the objective lenses together, but currently we move the sample.

Fig. 4
Fig. 4

Left panel shows the two PSF’s of Fig. 1 from two objective lenses at 60 deg. On the right, the two PSF’s were multiplied to give a confocal PSF that preserves the lateral resolution and provides an axial resolution nearly as good as the lateral. Again contours are at 5% and 50% of peak intensity.

Fig. 5
Fig. 5

Layout with which the images were taken. The Y direction is out of the paper and is effected by the galvanometer mirror, which is optically conjugate to the pupils of the objective lenses. The primary plane of the figure contains the galvanometer and the two objective lenses, as well as the turning mirrors M2 and M3 and the two field lenses L1 and L2. Turning mirrors M1 and M4 allow the incident laser beam and the exiting signal to come from slightly below the primary plane. Direction of the light is shown by the large arrows, whereas the small arrows indicate directions in which the objective lenses can move in the proposed final version. T′ and T″ are the points conjugate to the object T. T″ is used during the alignment process.

Fig. 6
Fig. 6

Scan line curvature in the XY plane that is due to the light’s incidence on the galvanometer mirror not being in a plane perpendicular to the galvanometer axis. We find ΔX to be 0.2 µm, so the curvature is negligible and the line is straight enough to use in alignment.

Fig. 7
Fig. 7

Curvature in Y, Z, displayed by imaging the surface of a cover slip in the Y, Z scan. The width of the field is 2.25 mm and the sag is 20 µm. This curvature is inherent in the design because the focal surface is a cylinder. It requires anamorphic optics to correct this, but some tricks are possible to minimize it, and it can be removed in the software if the task is three-dimensional imaging.

Fig. 8
Fig. 8

Air Force target in the X, Y scan. The smallest resolved group demonstrates 4.4-µm lateral resolution, as predicted.

Fig. 9
Fig. 9

ZY scan of the Air Force target at about the line indicated by the arrow in Fig. 8 displays the axial resolution of 4.5 µm. This figure spans 630 µm horizontally (Y) and 36 µm vertically (Z). The profile, taken from the actual pixel values, allows measurement of the resolution without the complication of visual saturation.

Fig. 10
Fig. 10

YZ section of skin at two intensities. The image on the right allows the epidermis and basal layer to saturate to show the depth of imaging into the dermis.

Fig. 11
Fig. 11

Profiles of averaged intensity for the two images of Fig. 10. The top graph is the unsaturated image, where the surface appears at approximately 16 µm and the basal layer at approximately 46 µm. The bottom graph is the dermis that shows the saturation of the epidermal region and the exponential decay of signal until it is lost in the noise at approximately 300 µm deep in the dermis.

Equations (3)

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Δr=0.44λ/NA,
Δz=1.5nλ/NA2,
Δx=d tan α1cos β-1.

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