Abstract

The non-invasive determination of deep tissue three dimensional structure and biochemistry is the ultimate goal of optical biopsy. Two-photon microscopy has been shown to be a particularly promising approach. The use of infrared radiation in two-photon microscopy is critical for deep tissue imaging since tissue absorption and scattering coefficients for infrared light are much lower than for shorter wavelengths. Equally important, tissue photodamage is localized to the focal region where fluorescence excitation occurs. This report demonstrates that, by means of high resolution two-photon microscopy, skin and subcutaneous tissue structures can be imaged utilizing their endogenous fluorescence. From a freshly prepared tissue punch of a mouse ear, we were able to resolve in 3D both the living and cornified keratinocytes in the epidermis, the collagen/elastin fibers in the dermal layer and the cartilage in the subcutaneous layer. The ability to non-invasively acquire 3D structures of these tissue components may find application in areas such as non-invasive diagnosis of skin cancer and the study of wound healing processes.

© Optical Society of America

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References

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  4. S. Robbins and M. Angell, Basic Pathology, (W. B. Saunders Co., Philadelphia, PA 1971).
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
  9. M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, and R. R. Anderson, "In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast," J. Investigative Dermatology 6, 946-954 (1995).
    [CrossRef]
  10. P. Corcuff, C. Bertrand, and L. Leveque, "Morphometry of human epidermis in vivo by real-time confocal Microscopy," Arch. Dermatol. Res 285, 475-481 (1993).
    [CrossRef] [PubMed]
  11. B. R. Masters, G., Gonnord, P. Corcuff, "Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope," J. Micros. 185, 329-338 (1997).
    [CrossRef]
  12. B. R. Masters, "Three-Dimensional Confocal Microscopy of Human Skin In Vivo: Autofluorescence of Normal Skin," Bioimages 4, 13-19, (1996).
  13. V. Rummelt, L. M. G. Gardner, R. Folberg, S. Beck, B. Knosp, T. O. Moninger, and K. C. Moore, "Three-dimensional relationship between tumor cells and micrcirculation with double cyanine immunolabelling, laser scanning confocal microscopy, and computer assisted reconstruction: an alternative to cast corrosion preparation," J. Histochemistry and cytochemistry 42, 681-686 (1994).
    [CrossRef]
  14. T. Wilson and C. Sheppard, Theory and practice of scanning optical microscopy, (Academic Press, New York, NY, 1984).
  15. J. B. Pawley, Handbook of Biological Confocal Microscopy,.(Plenum Press, New York, 1995).
    [CrossRef]
  16. B. R. Masters, "In vivo corneal redox fluorometry", in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990).
    [CrossRef]
  17. M. Göppert-Mayer, "über Elementarake mit zwei Quantensprungen," Ann. Phys. (Leipzig) 5, 273-294 (1931).
  18. M. J. Booth, S. W. Hell, "Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line," J. Microsc. 190, 298-304 (1998).
    [CrossRef] [PubMed]
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    [PubMed]
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    [CrossRef] [PubMed]
  23. B. R. Masters and B. Chance, "Redox confocal imaging: intrinsic fluorescent probes of cellular metabolism" in Fluorescent and Luminescent Probes for Biological Activity, W.T. Mason, ed., (Academic Press, London, 1993).
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    [CrossRef] [PubMed]
  25. W. Zipfel, "Multi-photon excitation of intrinsic fluorescence in cells and intact tissue", Presented in Application of multi-photon excitation imaging, Pre-Microscope Society of America Symposium, Cleveland, OH, Aug 9-10, 1997.
  26. F. S. LaBella, and P. Gerald, "Structure of collagen from human tendon as influence by age and sex," J. Gerontol. 20, 54-59 (1965).
    [PubMed]
  27. M. K. Dabbous, "Inter- and intramolecula cross-linking in tyrosinase-treated tropocollagen," J. Bio. Chem. 241, 5307-5312 (1966).
  28. K. C. Hoerman and A. Y. Balekjian, "Some quantum aspects of collagen," Federation Proc. 25, 1016- 1021 (1966).
  29. J. Thomas, D. F. Elsden, S. M. Partridge, "Degradation products from elastin," Nature 200, 651-652 (1963).
    [CrossRef] [PubMed]
  30. F. S. LeBella, "Studies on the soluble products released from purified elastic fibers by pancreatic elastase," Arch. Biochm. Biophys. 93, 72-79 (1961).
    [CrossRef]
  31. F. S. LaBella, and W. G. Lindsay, "The structure of human aortic elastin as influence by age," J. Gerontol. 18, 111-118, 1963.
    [PubMed]
  32. P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, "Time-resolved fluorescence microscopy using two-photon excitation," Bioimaging 3, 49-63 (1995).
    [CrossRef]

Other (32)

G. Murphy and D. Elder, Atlas of Tumor Pathology: Non-Melanocytic Tumors of the Skin, (Armed Forces Institute of Pathology, Washington, D.C., 1990).

D. Elder and G. Murphy, Atlas of Tumor Pathology: Melanocytic Tumors of the Skin, (Armed Forces Institute of Pathology, Washington, D.C. 1990).

M. Okun, Gross and Microscopic Pathology of the Skin, (Dermatopathology Foundation Press, Boston, 1976).

S. Robbins and M. Angell, Basic Pathology, (W. B. Saunders Co., Philadelphia, PA 1971).

R. A. F. Clark and P. M. Henson, The molecular and cellular biology of wound repair, (Plenum Press, New York, 1988).

W. Denk, W., J. H. Strickler and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

D. W. Piston, B. R. Masters, and W. W. Webb, "Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy," J. Micros. 178, 20-27, (1995).
[CrossRef]

B. R. Masters, P. T. C. So, and E. Gratton, "Multi-Photon Excitation Fluorescence Microscopy and Spectroscopy of In Vivo Human Skin," Biophys. J. 72, 2405-2412 (1997).
[CrossRef] [PubMed]

M. Raijadhyaksha, M. Grossman, D. Esterowitz, R. H. Webb, and R. R. Anderson, "In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast," J. Investigative Dermatology 6, 946-954 (1995).
[CrossRef]

P. Corcuff, C. Bertrand, and L. Leveque, "Morphometry of human epidermis in vivo by real-time confocal Microscopy," Arch. Dermatol. Res 285, 475-481 (1993).
[CrossRef] [PubMed]

B. R. Masters, G., Gonnord, P. Corcuff, "Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope," J. Micros. 185, 329-338 (1997).
[CrossRef]

B. R. Masters, "Three-Dimensional Confocal Microscopy of Human Skin In Vivo: Autofluorescence of Normal Skin," Bioimages 4, 13-19, (1996).

V. Rummelt, L. M. G. Gardner, R. Folberg, S. Beck, B. Knosp, T. O. Moninger, and K. C. Moore, "Three-dimensional relationship between tumor cells and micrcirculation with double cyanine immunolabelling, laser scanning confocal microscopy, and computer assisted reconstruction: an alternative to cast corrosion preparation," J. Histochemistry and cytochemistry 42, 681-686 (1994).
[CrossRef]

T. Wilson and C. Sheppard, Theory and practice of scanning optical microscopy, (Academic Press, New York, NY, 1984).

J. B. Pawley, Handbook of Biological Confocal Microscopy,.(Plenum Press, New York, 1995).
[CrossRef]

B. R. Masters, "In vivo corneal redox fluorometry", in Noninvasive Diagnostic Techniques in Ophthalmology, B. R. Masters, ed. (Springer-Verlag, New York, 1990).
[CrossRef]

M. Göppert-Mayer, "über Elementarake mit zwei Quantensprungen," Ann. Phys. (Leipzig) 5, 273-294 (1931).

M. J. Booth, S. W. Hell, "Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line," J. Microsc. 190, 298-304 (1998).
[CrossRef] [PubMed]

B. Chance, and B. Thorell, "Localization and kinetics of reduced pyridine nucleotide in living cells by Microfluorometry," J. Biol. Chem. 234, 3044-3050 (1959).
[PubMed]

B. Chance, "Pyridine nucleotide as an indicator of the oxygen requirements for energy-linked functions of Mitochondria," Circ. Res. Suppl. 1 38, I-31 - I-38 (1976).

B. Chance, B. Schoener, R. Oshino, F. Itshak, & Y. Nakase, "Oxidation-Reduction Ratio Studies of Mitochondria in Freeze-trapped Samples," J. Biol. Chem. 254, 4764-4711 (1979).
[PubMed]

B. D. Bennett, T. L. Jetton, G. Ying, M. A. Magnuson and D. W. Piston, "Quantitative Subcellular Imaging of Glucose Metabolism within Intact Pancreatic Islets," J. Biol. Chem. 271, 3647-3651 (1996).
[CrossRef] [PubMed]

B. R. Masters and B. Chance, "Redox confocal imaging: intrinsic fluorescent probes of cellular metabolism" in Fluorescent and Luminescent Probes for Biological Activity, W.T. Mason, ed., (Academic Press, London, 1993).

S. Maiti, J. B. Shear, R. M. Williams RM, W. R. Zipfel, W. W. Webb, "Measuring serotonin distribution in live cells with three-photon excitation," Science 275, 530-532 (1997).
[CrossRef] [PubMed]

W. Zipfel, "Multi-photon excitation of intrinsic fluorescence in cells and intact tissue", Presented in Application of multi-photon excitation imaging, Pre-Microscope Society of America Symposium, Cleveland, OH, Aug 9-10, 1997.

F. S. LaBella, and P. Gerald, "Structure of collagen from human tendon as influence by age and sex," J. Gerontol. 20, 54-59 (1965).
[PubMed]

M. K. Dabbous, "Inter- and intramolecula cross-linking in tyrosinase-treated tropocollagen," J. Bio. Chem. 241, 5307-5312 (1966).

K. C. Hoerman and A. Y. Balekjian, "Some quantum aspects of collagen," Federation Proc. 25, 1016- 1021 (1966).

J. Thomas, D. F. Elsden, S. M. Partridge, "Degradation products from elastin," Nature 200, 651-652 (1963).
[CrossRef] [PubMed]

F. S. LeBella, "Studies on the soluble products released from purified elastic fibers by pancreatic elastase," Arch. Biochm. Biophys. 93, 72-79 (1961).
[CrossRef]

F. S. LaBella, and W. G. Lindsay, "The structure of human aortic elastin as influence by age," J. Gerontol. 18, 111-118, 1963.
[PubMed]

P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, and E. Gratton, "Time-resolved fluorescence microscopy using two-photon excitation," Bioimaging 3, 49-63 (1995).
[CrossRef]

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Figures (6)

Fig. 1.
Fig. 1.

A comparison between two-photon and confocal microscope detection geometry. In a highly scattering medium, a large fraction of emitted photons will be scattered before they are collected by the objective. In the two-photon system, most of these scattered photons can be collected by a large area detector. In the confocal system, these scattered photons are blocked at the confocal pinhole aperture and cannot be detected.

Fig. 2.
Fig. 2.

Schematic of the prototype two-photon deep tissue microscope

Fig. 3.
Fig. 3.

Corss section of mouse ear (one-half of full thickness). The presence of a cartilage layer below the skin is unique to the ear.

Fig. 4.
Fig. 4.

A 3-D reconstructed movie sequence showing the relevant dermal and subcutaneous structures of the mouse tissue punch as imaged by two-photon microscopy. [Media 1]

Fig. 5.
Fig. 5.

A montage of x-y sections of mouse ear structures obtained by two-photon deep tissue microscopy. From left to right, the five panels are images of: stratum corneum, epidermal cell layer, basal cell layer, dermal structure, and cartilage. The depth where these images were taken are indicated in the upper left corner of the panel. The scale bar is 20 μm.

Fig. 6.
Fig. 6.

Light microscopy image of a histological section of the mouse ear tissue punch. The scale bar at the lower left corner represents 50 μm. S is the stratum corneum, E is the epidermal cell layers. B is the basal cell layer, D is the dermal layer, and C is the cartilage. The blue arrows indicate tissue tears common in the preparation of histological section.

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