Abstract

We discuss the design and construction of a novel imaging system in which a fiber-optic imaging bundle and miniature optical and mechanical components are used to allow confocal fluorescence microscopy in remote locations. The instrumentation has been developed specifically for cellular examination of tissue for optical biopsy. Miniaturization of various components makes the device usable in a clinical setting. The numerical aperture of the beam in the tissue is 0.5, and the field of view is 430 µm. The measured lateral resolution of the system is 3.0 µm. The axial point and the axial planar response functions of the confocal system were measured with a FWHM of 10 and 25 µm, respectively. In vitro and in vivo images obtained with cell cultures, human tissue specimens, and animal models indicate that the performance of the device is adequate for microscopic evaluation of cells.

© 1999 Optical Society of America

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  1. M. Rajadhyaksha, R. G. B. Langley, S. Gonzales, M. White, J. M. Zavislan, R. H. Webb, R. R. Anderson, “Clinical real-time confocal imaging of human skin and oral tissues in vivo,” in Conference Proceedings of LEOS ’97, 10th Annual Meeting of the IEEE Lasers and Electro-Optics Society (IEEE, N.Y., 1997).
  2. M. Hammer, D. Schweitzer, L. Leistritz, M. Scibor, K. Donnerhacke, J. Strobel, “Imaging spectroscopy of human ocular fundus in vivo,” J. Biomed. Opt. 2, 418–425 (1997).
    [CrossRef] [PubMed]
  3. B. R. Masters, “Three-dimensional confocal microscopy of the living in situ rabbit cornea,” wOpt. Express 3, 59–63 (1998), http://epubs.osa.org/opticsexpress .
    [CrossRef]
  4. A. F. Gmitro, D. Aziz, “Confocal microscopy through a fiber-optic imaging bundle,” Opt. Lett. 18, 565–567 (1993).
    [CrossRef] [PubMed]
  5. R. Juskaitis, T. Wilson, T. F. Watson, “Real-time white light reflection confocal microscopy using a fibre-optic bundle,” Scanning 19, 15–19 (1997).
    [CrossRef]
  6. T. Collier, C. Smithpeter, B. Cowman, R. Drezek, M. Bescour, R. Richards-Kortum, “Fiber-optic confocal microscope for biological imaging,” in Conference on Lasers and Electro-Optics, Vol. 6 of 1998 Technical Digest Series (Optical Society of America, Washington, D.C., 1998), pp. 128–129.
  7. D. L. Dickensheets, G. S. Kino, “Silicon-micromachined scanning confocal optical microscope,” J. Microelectromech. Syst. 7, 38–47 (1998).
    [CrossRef]
  8. M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467 (1961).
  9. D. Aziz, “Confocal microscopy through a fiber-optic imaging bundle,” Ph.D. dissertation (Optical Sciences Library, The University of Arizona, Tucson, Ariz., 1994), pp. 25–30.
  10. Y. Sabharwal, “Remote-access slit-scanning confocal microscope for in vivo tumor diagnosis,” Ph.D. dissertation (Optical Sciences Library, The University of Arizona, Tucson, Ariz., 1998), pp. 50–51.
  11. T. Wilson, Confocal Microscopy (Academic, London, 1990), pp. 114–118.
  12. M. A. Player, “Spread functions and modulation transfer functions of fibre-optic bundles,” J. Mod. Opt. 35, 1363–1372 (1988).
    [CrossRef]
  13. Y. Sabharwal, “Remote-access slit-scanning confocal microscope for in vivo tumor diagnosis,” Ph.D. dissertation (Optical Sciences Library, (The University of Arizona, Tucson, Ariz., 1998), pp. 51–54.
  14. J. D. Gaskill, Linear Systems, Fourier Transforms, and Optics (Wiley, New York, 1978), pp. 339–345.
  15. R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Inc., Eugene, Oreg., 1996), pp. 148–149.
  16. A. R. Parrish, A. J. Gandolfi, K. Brendel, “Precision-cut tissue slices: applications in pharmacology and toxicology,” Life Sci. 57, 1887–1901 (1995).
    [CrossRef] [PubMed]

1998 (2)

D. L. Dickensheets, G. S. Kino, “Silicon-micromachined scanning confocal optical microscope,” J. Microelectromech. Syst. 7, 38–47 (1998).
[CrossRef]

B. R. Masters, “Three-dimensional confocal microscopy of the living in situ rabbit cornea,” wOpt. Express 3, 59–63 (1998), http://epubs.osa.org/opticsexpress .
[CrossRef]

1997 (2)

M. Hammer, D. Schweitzer, L. Leistritz, M. Scibor, K. Donnerhacke, J. Strobel, “Imaging spectroscopy of human ocular fundus in vivo,” J. Biomed. Opt. 2, 418–425 (1997).
[CrossRef] [PubMed]

R. Juskaitis, T. Wilson, T. F. Watson, “Real-time white light reflection confocal microscopy using a fibre-optic bundle,” Scanning 19, 15–19 (1997).
[CrossRef]

1995 (1)

A. R. Parrish, A. J. Gandolfi, K. Brendel, “Precision-cut tissue slices: applications in pharmacology and toxicology,” Life Sci. 57, 1887–1901 (1995).
[CrossRef] [PubMed]

1993 (1)

1988 (1)

M. A. Player, “Spread functions and modulation transfer functions of fibre-optic bundles,” J. Mod. Opt. 35, 1363–1372 (1988).
[CrossRef]

Anderson, R. R.

M. Rajadhyaksha, R. G. B. Langley, S. Gonzales, M. White, J. M. Zavislan, R. H. Webb, R. R. Anderson, “Clinical real-time confocal imaging of human skin and oral tissues in vivo,” in Conference Proceedings of LEOS ’97, 10th Annual Meeting of the IEEE Lasers and Electro-Optics Society (IEEE, N.Y., 1997).

Aziz, D.

A. F. Gmitro, D. Aziz, “Confocal microscopy through a fiber-optic imaging bundle,” Opt. Lett. 18, 565–567 (1993).
[CrossRef] [PubMed]

D. Aziz, “Confocal microscopy through a fiber-optic imaging bundle,” Ph.D. dissertation (Optical Sciences Library, The University of Arizona, Tucson, Ariz., 1994), pp. 25–30.

Bescour, M.

T. Collier, C. Smithpeter, B. Cowman, R. Drezek, M. Bescour, R. Richards-Kortum, “Fiber-optic confocal microscope for biological imaging,” in Conference on Lasers and Electro-Optics, Vol. 6 of 1998 Technical Digest Series (Optical Society of America, Washington, D.C., 1998), pp. 128–129.

Brendel, K.

A. R. Parrish, A. J. Gandolfi, K. Brendel, “Precision-cut tissue slices: applications in pharmacology and toxicology,” Life Sci. 57, 1887–1901 (1995).
[CrossRef] [PubMed]

Collier, T.

T. Collier, C. Smithpeter, B. Cowman, R. Drezek, M. Bescour, R. Richards-Kortum, “Fiber-optic confocal microscope for biological imaging,” in Conference on Lasers and Electro-Optics, Vol. 6 of 1998 Technical Digest Series (Optical Society of America, Washington, D.C., 1998), pp. 128–129.

Cowman, B.

T. Collier, C. Smithpeter, B. Cowman, R. Drezek, M. Bescour, R. Richards-Kortum, “Fiber-optic confocal microscope for biological imaging,” in Conference on Lasers and Electro-Optics, Vol. 6 of 1998 Technical Digest Series (Optical Society of America, Washington, D.C., 1998), pp. 128–129.

Dickensheets, D. L.

D. L. Dickensheets, G. S. Kino, “Silicon-micromachined scanning confocal optical microscope,” J. Microelectromech. Syst. 7, 38–47 (1998).
[CrossRef]

Donnerhacke, K.

M. Hammer, D. Schweitzer, L. Leistritz, M. Scibor, K. Donnerhacke, J. Strobel, “Imaging spectroscopy of human ocular fundus in vivo,” J. Biomed. Opt. 2, 418–425 (1997).
[CrossRef] [PubMed]

Drezek, R.

T. Collier, C. Smithpeter, B. Cowman, R. Drezek, M. Bescour, R. Richards-Kortum, “Fiber-optic confocal microscope for biological imaging,” in Conference on Lasers and Electro-Optics, Vol. 6 of 1998 Technical Digest Series (Optical Society of America, Washington, D.C., 1998), pp. 128–129.

Gandolfi, A. J.

A. R. Parrish, A. J. Gandolfi, K. Brendel, “Precision-cut tissue slices: applications in pharmacology and toxicology,” Life Sci. 57, 1887–1901 (1995).
[CrossRef] [PubMed]

Gaskill, J. D.

J. D. Gaskill, Linear Systems, Fourier Transforms, and Optics (Wiley, New York, 1978), pp. 339–345.

Gmitro, A. F.

Gonzales, S.

M. Rajadhyaksha, R. G. B. Langley, S. Gonzales, M. White, J. M. Zavislan, R. H. Webb, R. R. Anderson, “Clinical real-time confocal imaging of human skin and oral tissues in vivo,” in Conference Proceedings of LEOS ’97, 10th Annual Meeting of the IEEE Lasers and Electro-Optics Society (IEEE, N.Y., 1997).

Hammer, M.

M. Hammer, D. Schweitzer, L. Leistritz, M. Scibor, K. Donnerhacke, J. Strobel, “Imaging spectroscopy of human ocular fundus in vivo,” J. Biomed. Opt. 2, 418–425 (1997).
[CrossRef] [PubMed]

Haugland, R. P.

R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Inc., Eugene, Oreg., 1996), pp. 148–149.

Juskaitis, R.

R. Juskaitis, T. Wilson, T. F. Watson, “Real-time white light reflection confocal microscopy using a fibre-optic bundle,” Scanning 19, 15–19 (1997).
[CrossRef]

Kino, G. S.

D. L. Dickensheets, G. S. Kino, “Silicon-micromachined scanning confocal optical microscope,” J. Microelectromech. Syst. 7, 38–47 (1998).
[CrossRef]

Langley, R. G. B.

M. Rajadhyaksha, R. G. B. Langley, S. Gonzales, M. White, J. M. Zavislan, R. H. Webb, R. R. Anderson, “Clinical real-time confocal imaging of human skin and oral tissues in vivo,” in Conference Proceedings of LEOS ’97, 10th Annual Meeting of the IEEE Lasers and Electro-Optics Society (IEEE, N.Y., 1997).

Leistritz, L.

M. Hammer, D. Schweitzer, L. Leistritz, M. Scibor, K. Donnerhacke, J. Strobel, “Imaging spectroscopy of human ocular fundus in vivo,” J. Biomed. Opt. 2, 418–425 (1997).
[CrossRef] [PubMed]

Masters, B. R.

B. R. Masters, “Three-dimensional confocal microscopy of the living in situ rabbit cornea,” wOpt. Express 3, 59–63 (1998), http://epubs.osa.org/opticsexpress .
[CrossRef]

Minsky, M.

M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467 (1961).

Parrish, A. R.

A. R. Parrish, A. J. Gandolfi, K. Brendel, “Precision-cut tissue slices: applications in pharmacology and toxicology,” Life Sci. 57, 1887–1901 (1995).
[CrossRef] [PubMed]

Player, M. A.

M. A. Player, “Spread functions and modulation transfer functions of fibre-optic bundles,” J. Mod. Opt. 35, 1363–1372 (1988).
[CrossRef]

Rajadhyaksha, M.

M. Rajadhyaksha, R. G. B. Langley, S. Gonzales, M. White, J. M. Zavislan, R. H. Webb, R. R. Anderson, “Clinical real-time confocal imaging of human skin and oral tissues in vivo,” in Conference Proceedings of LEOS ’97, 10th Annual Meeting of the IEEE Lasers and Electro-Optics Society (IEEE, N.Y., 1997).

Richards-Kortum, R.

T. Collier, C. Smithpeter, B. Cowman, R. Drezek, M. Bescour, R. Richards-Kortum, “Fiber-optic confocal microscope for biological imaging,” in Conference on Lasers and Electro-Optics, Vol. 6 of 1998 Technical Digest Series (Optical Society of America, Washington, D.C., 1998), pp. 128–129.

Sabharwal, Y.

Y. Sabharwal, “Remote-access slit-scanning confocal microscope for in vivo tumor diagnosis,” Ph.D. dissertation (Optical Sciences Library, (The University of Arizona, Tucson, Ariz., 1998), pp. 51–54.

Y. Sabharwal, “Remote-access slit-scanning confocal microscope for in vivo tumor diagnosis,” Ph.D. dissertation (Optical Sciences Library, The University of Arizona, Tucson, Ariz., 1998), pp. 50–51.

Schweitzer, D.

M. Hammer, D. Schweitzer, L. Leistritz, M. Scibor, K. Donnerhacke, J. Strobel, “Imaging spectroscopy of human ocular fundus in vivo,” J. Biomed. Opt. 2, 418–425 (1997).
[CrossRef] [PubMed]

Scibor, M.

M. Hammer, D. Schweitzer, L. Leistritz, M. Scibor, K. Donnerhacke, J. Strobel, “Imaging spectroscopy of human ocular fundus in vivo,” J. Biomed. Opt. 2, 418–425 (1997).
[CrossRef] [PubMed]

Smithpeter, C.

T. Collier, C. Smithpeter, B. Cowman, R. Drezek, M. Bescour, R. Richards-Kortum, “Fiber-optic confocal microscope for biological imaging,” in Conference on Lasers and Electro-Optics, Vol. 6 of 1998 Technical Digest Series (Optical Society of America, Washington, D.C., 1998), pp. 128–129.

Strobel, J.

M. Hammer, D. Schweitzer, L. Leistritz, M. Scibor, K. Donnerhacke, J. Strobel, “Imaging spectroscopy of human ocular fundus in vivo,” J. Biomed. Opt. 2, 418–425 (1997).
[CrossRef] [PubMed]

Watson, T. F.

R. Juskaitis, T. Wilson, T. F. Watson, “Real-time white light reflection confocal microscopy using a fibre-optic bundle,” Scanning 19, 15–19 (1997).
[CrossRef]

Webb, R. H.

M. Rajadhyaksha, R. G. B. Langley, S. Gonzales, M. White, J. M. Zavislan, R. H. Webb, R. R. Anderson, “Clinical real-time confocal imaging of human skin and oral tissues in vivo,” in Conference Proceedings of LEOS ’97, 10th Annual Meeting of the IEEE Lasers and Electro-Optics Society (IEEE, N.Y., 1997).

White, M.

M. Rajadhyaksha, R. G. B. Langley, S. Gonzales, M. White, J. M. Zavislan, R. H. Webb, R. R. Anderson, “Clinical real-time confocal imaging of human skin and oral tissues in vivo,” in Conference Proceedings of LEOS ’97, 10th Annual Meeting of the IEEE Lasers and Electro-Optics Society (IEEE, N.Y., 1997).

Wilson, T.

R. Juskaitis, T. Wilson, T. F. Watson, “Real-time white light reflection confocal microscopy using a fibre-optic bundle,” Scanning 19, 15–19 (1997).
[CrossRef]

T. Wilson, Confocal Microscopy (Academic, London, 1990), pp. 114–118.

Zavislan, J. M.

M. Rajadhyaksha, R. G. B. Langley, S. Gonzales, M. White, J. M. Zavislan, R. H. Webb, R. R. Anderson, “Clinical real-time confocal imaging of human skin and oral tissues in vivo,” in Conference Proceedings of LEOS ’97, 10th Annual Meeting of the IEEE Lasers and Electro-Optics Society (IEEE, N.Y., 1997).

J. Biomed. Opt. (1)

M. Hammer, D. Schweitzer, L. Leistritz, M. Scibor, K. Donnerhacke, J. Strobel, “Imaging spectroscopy of human ocular fundus in vivo,” J. Biomed. Opt. 2, 418–425 (1997).
[CrossRef] [PubMed]

J. Microelectromech. Syst. (1)

D. L. Dickensheets, G. S. Kino, “Silicon-micromachined scanning confocal optical microscope,” J. Microelectromech. Syst. 7, 38–47 (1998).
[CrossRef]

J. Mod. Opt. (1)

M. A. Player, “Spread functions and modulation transfer functions of fibre-optic bundles,” J. Mod. Opt. 35, 1363–1372 (1988).
[CrossRef]

Life Sci. (1)

A. R. Parrish, A. J. Gandolfi, K. Brendel, “Precision-cut tissue slices: applications in pharmacology and toxicology,” Life Sci. 57, 1887–1901 (1995).
[CrossRef] [PubMed]

Opt. Lett. (1)

Scanning (1)

R. Juskaitis, T. Wilson, T. F. Watson, “Real-time white light reflection confocal microscopy using a fibre-optic bundle,” Scanning 19, 15–19 (1997).
[CrossRef]

wOpt. Express (1)

B. R. Masters, “Three-dimensional confocal microscopy of the living in situ rabbit cornea,” wOpt. Express 3, 59–63 (1998), http://epubs.osa.org/opticsexpress .
[CrossRef]

Other (9)

T. Collier, C. Smithpeter, B. Cowman, R. Drezek, M. Bescour, R. Richards-Kortum, “Fiber-optic confocal microscope for biological imaging,” in Conference on Lasers and Electro-Optics, Vol. 6 of 1998 Technical Digest Series (Optical Society of America, Washington, D.C., 1998), pp. 128–129.

M. Rajadhyaksha, R. G. B. Langley, S. Gonzales, M. White, J. M. Zavislan, R. H. Webb, R. R. Anderson, “Clinical real-time confocal imaging of human skin and oral tissues in vivo,” in Conference Proceedings of LEOS ’97, 10th Annual Meeting of the IEEE Lasers and Electro-Optics Society (IEEE, N.Y., 1997).

Y. Sabharwal, “Remote-access slit-scanning confocal microscope for in vivo tumor diagnosis,” Ph.D. dissertation (Optical Sciences Library, (The University of Arizona, Tucson, Ariz., 1998), pp. 51–54.

J. D. Gaskill, Linear Systems, Fourier Transforms, and Optics (Wiley, New York, 1978), pp. 339–345.

R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Inc., Eugene, Oreg., 1996), pp. 148–149.

M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467 (1961).

D. Aziz, “Confocal microscopy through a fiber-optic imaging bundle,” Ph.D. dissertation (Optical Sciences Library, The University of Arizona, Tucson, Ariz., 1994), pp. 25–30.

Y. Sabharwal, “Remote-access slit-scanning confocal microscope for in vivo tumor diagnosis,” Ph.D. dissertation (Optical Sciences Library, The University of Arizona, Tucson, Ariz., 1998), pp. 50–51.

T. Wilson, Confocal Microscopy (Academic, London, 1990), pp. 114–118.

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Figures (17)

Fig. 1
Fig. 1

Layout of the remote-access, slit-scanning confocal microscope system (patent pending).

Fig. 2
Fig. 2

Miniature objective lens design (patent pending).

Fig. 3
Fig. 3

Polychromatic MTF plot of miniature objective lens.

Fig. 4
Fig. 4

Schematic of focus and control subassemblies of miniature positioning mechanism (patent pending).

Fig. 5
Fig. 5

Objective lens and subassembly of focusing mechanism.

Fig. 6
Fig. 6

Illumination LSF at proximal and distal fiber faces.

Fig. 7
Fig. 7

Effective fiber-bundle PSF.

Fig. 8
Fig. 8

Bar target images obtained (a) without and (b) with the objective lens.

Fig. 9
Fig. 9

Measured MTF of the miniature objective lens.

Fig. 10
Fig. 10

(a) Measured average edge function, (b) LSF calculated with the measured edge function.

Fig. 11
Fig. 11

Comparison of measured and simulated axial point response functions for confocal system with the objective lens and fiber bundle.

Fig. 12
Fig. 12

Comparison of measured and simulated axial planar response functions for a confocal system with the objective lens and fiber bundle.

Fig. 13
Fig. 13

Fluorescence images obtained with the confocal microendoscope: (a) 15-µm fluorescent microspheres; (b) 6-µm fluorescent microspheres.

Fig. 14
Fig. 14

Image of PC3 cells in culture stained with SYTO-16 and acquired with the confocal microendoscope.

Fig. 15
Fig. 15

Images of normal human prostate tissue taken at various depths: (a) tissue surface; (b) 15 µm below tissue surface; (c) 30 µm below tissue surface; (d) 45 µm below tissue surface.

Fig. 16
Fig. 16

Images of abnormal human prostate tissue: (a) indications of hyperplasia; (b) region of aggressive cancer.

Fig. 17
Fig. 17

In vivo images of a mouse: (a) top surface of peritoneal lining; (b) underlying peritoneal and smooth muscle cells.

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