Abstract

We describe a holographic microscope with a spatial resolution approaching the diffraction limit. The instrument uses a tiny drop of glycerol as a lens to create the spherically diverging reference illumination necessary for Fourier-transform holography. Measurement of the point-spread function, which is obtained by imaging a knife edge in dark-field illumination, indicates a transverse resolution of 1.4 μm with wavelength λ = 514.5 nm. Longitudinal resolution is obtained from the holograms by the numerical equivalent of optical sectioning. We describe the method of reconstruction and demonstrate the microscope’s capability with selected biological specimens. The instrument offers two unique capabilities: (1) it can collect three-dimensional information in a single pulse of light, avoiding specimen damage and bleaching; and (2) it can record three-dimensional motion pictures from a series of light pulses. The conceptual design is applicable to a broad range of wavelengths and we discuss extension to the x-ray regime.

© 1992 Optical Society of America

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  1. D. Gabor, “A new microscopic principle,” Nature 161, 777–778 (1948); Proc. R. Soc. London Ser. A 197, 454–487 (1949).
    [CrossRef] [PubMed]
  2. G. Stroke, “Attainment of high resolutions in image-forming x-ray microscopy with ‘lensless’ Fourier-transform holograms and correlative source-effect compensation,” in Optique des Rayons X et Microanalyse (Hermann, Paris, 1966), pp. 30–46.
  3. J. Winthrop, C. Worthington, “X-ray microscopy by successive Fourier transformation,” Phys. Lett. 15, 124–126 (1965); G. Stroke, R. Restrick, “Holography with spatially noncoherent light,” Appl. Phys. Lett. 7, 229–230 (1966); G. Stroke, D. Falconer, “Attainment of high resolutions in wavefront reconstruction imaging—II,” J. Opt. Soc. Am. 55, 595 (1965).
    [CrossRef]
  4. E. Leith, J. Upatnieks, “Microscopy by wavefront reconstruction,” J. Opt. Soc. Am. 55, 569–570 (1965); E. Leith, J. Upatnieks, A. VanderLugt, “Hologram microscopy and lens aberration compensation by the use of holograms,” J. Opt. Soc. Am. 55, 595 (1965).
    [CrossRef]
  5. R. VanLigten, H. Osterberg, “Holographic microscopy,” Nature 211, 282–283 (1966).
    [CrossRef]
  6. J. C. Solem, G. C. Baldwin, G. F. Chapline, “Holography at x-ray wavelengths,” in Proceedings of International Conference on Lasers 1981, C. B. Collins, ed. (STS, McClean, Va, 1981), pp. 296–235; J. C. Solem, G. C. Baldwin, “Microholography of living organisms,” Science 218, 229–235 (1982); J. C. Solem, G. F. Chapline, “X-ray biomicroholography,” Opt. Eng. 23, 193–203 (1984); I. McNulty, J. Kirz, C. Jacobsen, M. R. Howells, E. H. Anderson, “First results with a Fourier transform holographic microscope,” in X-Ray Microscopy III, A. G. Michette, G. R. Morrison, C. J. Buckley, eds. (Springer-Verlag, Berlin, 1992), 251–254.
    [CrossRef] [PubMed]
  7. W. S. Haddad, D. Cullen, K. Boyer, C. K. Rhodes, J. C. Solem, R. S. Weinstein, “Design for a Fourier-transform holographic microscope,” in X-Ray Microscopy II, D. Sayre, M. Howells, J. Kirz, H. Rarback, eds. (Springer-Verlag, Berlin, 1988), pp. 284–287; W. S. Haddad, J. C. Solem, D. Cullen, K. Boyer, C. K. Rhodes, “A description of the theory and apparatus for digital reconstruction of Fourier transform holograms,” in Electronics Imaging ‘87, advance printing of paper summaries (Institute for Graphic Communication, Boston, Mass., 1987), Vol. II, pp. 683–688; W. S. Haddad, D. Cullen, J. C. Solem, K. Boyer, C. K. Rhodes, “X-ray Fourier-transform holographic microscope,” in Short Wavelength Coherent Radiation: Generation and Applications, R. W. Falcone, J. Kirz, eds., Vol. 2 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1988), pp. 284–289.
  8. F. A. Jenkins, H. E. White, in Fundamentals of Optics, 4th ed. (McGraw-Hill, New York, 1976), pp. 71–72.
  9. H. T. M. van der Voort, G. J. Brakenhoff, “3-D image formation in high-aperture fluorescence confocal microscopy: a numerical analysis,” J. Microsc. (Oxford) 158, 43–54 (1989).
    [CrossRef]
  10. S. F. Gibson, F. Lanni, “Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy,” J. Opt. Soc. Am. A 8, 1601–1613 (1991).
    [CrossRef]

1991

1989

H. T. M. van der Voort, G. J. Brakenhoff, “3-D image formation in high-aperture fluorescence confocal microscopy: a numerical analysis,” J. Microsc. (Oxford) 158, 43–54 (1989).
[CrossRef]

1966

R. VanLigten, H. Osterberg, “Holographic microscopy,” Nature 211, 282–283 (1966).
[CrossRef]

1965

J. Winthrop, C. Worthington, “X-ray microscopy by successive Fourier transformation,” Phys. Lett. 15, 124–126 (1965); G. Stroke, R. Restrick, “Holography with spatially noncoherent light,” Appl. Phys. Lett. 7, 229–230 (1966); G. Stroke, D. Falconer, “Attainment of high resolutions in wavefront reconstruction imaging—II,” J. Opt. Soc. Am. 55, 595 (1965).
[CrossRef]

E. Leith, J. Upatnieks, “Microscopy by wavefront reconstruction,” J. Opt. Soc. Am. 55, 569–570 (1965); E. Leith, J. Upatnieks, A. VanderLugt, “Hologram microscopy and lens aberration compensation by the use of holograms,” J. Opt. Soc. Am. 55, 595 (1965).
[CrossRef]

1948

D. Gabor, “A new microscopic principle,” Nature 161, 777–778 (1948); Proc. R. Soc. London Ser. A 197, 454–487 (1949).
[CrossRef] [PubMed]

Baldwin, G. C.

J. C. Solem, G. C. Baldwin, G. F. Chapline, “Holography at x-ray wavelengths,” in Proceedings of International Conference on Lasers 1981, C. B. Collins, ed. (STS, McClean, Va, 1981), pp. 296–235; J. C. Solem, G. C. Baldwin, “Microholography of living organisms,” Science 218, 229–235 (1982); J. C. Solem, G. F. Chapline, “X-ray biomicroholography,” Opt. Eng. 23, 193–203 (1984); I. McNulty, J. Kirz, C. Jacobsen, M. R. Howells, E. H. Anderson, “First results with a Fourier transform holographic microscope,” in X-Ray Microscopy III, A. G. Michette, G. R. Morrison, C. J. Buckley, eds. (Springer-Verlag, Berlin, 1992), 251–254.
[CrossRef] [PubMed]

Boyer, K.

W. S. Haddad, D. Cullen, K. Boyer, C. K. Rhodes, J. C. Solem, R. S. Weinstein, “Design for a Fourier-transform holographic microscope,” in X-Ray Microscopy II, D. Sayre, M. Howells, J. Kirz, H. Rarback, eds. (Springer-Verlag, Berlin, 1988), pp. 284–287; W. S. Haddad, J. C. Solem, D. Cullen, K. Boyer, C. K. Rhodes, “A description of the theory and apparatus for digital reconstruction of Fourier transform holograms,” in Electronics Imaging ‘87, advance printing of paper summaries (Institute for Graphic Communication, Boston, Mass., 1987), Vol. II, pp. 683–688; W. S. Haddad, D. Cullen, J. C. Solem, K. Boyer, C. K. Rhodes, “X-ray Fourier-transform holographic microscope,” in Short Wavelength Coherent Radiation: Generation and Applications, R. W. Falcone, J. Kirz, eds., Vol. 2 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1988), pp. 284–289.

Brakenhoff, G. J.

H. T. M. van der Voort, G. J. Brakenhoff, “3-D image formation in high-aperture fluorescence confocal microscopy: a numerical analysis,” J. Microsc. (Oxford) 158, 43–54 (1989).
[CrossRef]

Chapline, G. F.

J. C. Solem, G. C. Baldwin, G. F. Chapline, “Holography at x-ray wavelengths,” in Proceedings of International Conference on Lasers 1981, C. B. Collins, ed. (STS, McClean, Va, 1981), pp. 296–235; J. C. Solem, G. C. Baldwin, “Microholography of living organisms,” Science 218, 229–235 (1982); J. C. Solem, G. F. Chapline, “X-ray biomicroholography,” Opt. Eng. 23, 193–203 (1984); I. McNulty, J. Kirz, C. Jacobsen, M. R. Howells, E. H. Anderson, “First results with a Fourier transform holographic microscope,” in X-Ray Microscopy III, A. G. Michette, G. R. Morrison, C. J. Buckley, eds. (Springer-Verlag, Berlin, 1992), 251–254.
[CrossRef] [PubMed]

Cullen, D.

W. S. Haddad, D. Cullen, K. Boyer, C. K. Rhodes, J. C. Solem, R. S. Weinstein, “Design for a Fourier-transform holographic microscope,” in X-Ray Microscopy II, D. Sayre, M. Howells, J. Kirz, H. Rarback, eds. (Springer-Verlag, Berlin, 1988), pp. 284–287; W. S. Haddad, J. C. Solem, D. Cullen, K. Boyer, C. K. Rhodes, “A description of the theory and apparatus for digital reconstruction of Fourier transform holograms,” in Electronics Imaging ‘87, advance printing of paper summaries (Institute for Graphic Communication, Boston, Mass., 1987), Vol. II, pp. 683–688; W. S. Haddad, D. Cullen, J. C. Solem, K. Boyer, C. K. Rhodes, “X-ray Fourier-transform holographic microscope,” in Short Wavelength Coherent Radiation: Generation and Applications, R. W. Falcone, J. Kirz, eds., Vol. 2 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1988), pp. 284–289.

Gabor, D.

D. Gabor, “A new microscopic principle,” Nature 161, 777–778 (1948); Proc. R. Soc. London Ser. A 197, 454–487 (1949).
[CrossRef] [PubMed]

Gibson, S. F.

Haddad, W. S.

W. S. Haddad, D. Cullen, K. Boyer, C. K. Rhodes, J. C. Solem, R. S. Weinstein, “Design for a Fourier-transform holographic microscope,” in X-Ray Microscopy II, D. Sayre, M. Howells, J. Kirz, H. Rarback, eds. (Springer-Verlag, Berlin, 1988), pp. 284–287; W. S. Haddad, J. C. Solem, D. Cullen, K. Boyer, C. K. Rhodes, “A description of the theory and apparatus for digital reconstruction of Fourier transform holograms,” in Electronics Imaging ‘87, advance printing of paper summaries (Institute for Graphic Communication, Boston, Mass., 1987), Vol. II, pp. 683–688; W. S. Haddad, D. Cullen, J. C. Solem, K. Boyer, C. K. Rhodes, “X-ray Fourier-transform holographic microscope,” in Short Wavelength Coherent Radiation: Generation and Applications, R. W. Falcone, J. Kirz, eds., Vol. 2 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1988), pp. 284–289.

Jenkins, F. A.

F. A. Jenkins, H. E. White, in Fundamentals of Optics, 4th ed. (McGraw-Hill, New York, 1976), pp. 71–72.

Lanni, F.

Leith, E.

Osterberg, H.

R. VanLigten, H. Osterberg, “Holographic microscopy,” Nature 211, 282–283 (1966).
[CrossRef]

Rhodes, C. K.

W. S. Haddad, D. Cullen, K. Boyer, C. K. Rhodes, J. C. Solem, R. S. Weinstein, “Design for a Fourier-transform holographic microscope,” in X-Ray Microscopy II, D. Sayre, M. Howells, J. Kirz, H. Rarback, eds. (Springer-Verlag, Berlin, 1988), pp. 284–287; W. S. Haddad, J. C. Solem, D. Cullen, K. Boyer, C. K. Rhodes, “A description of the theory and apparatus for digital reconstruction of Fourier transform holograms,” in Electronics Imaging ‘87, advance printing of paper summaries (Institute for Graphic Communication, Boston, Mass., 1987), Vol. II, pp. 683–688; W. S. Haddad, D. Cullen, J. C. Solem, K. Boyer, C. K. Rhodes, “X-ray Fourier-transform holographic microscope,” in Short Wavelength Coherent Radiation: Generation and Applications, R. W. Falcone, J. Kirz, eds., Vol. 2 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1988), pp. 284–289.

Solem, J. C.

W. S. Haddad, D. Cullen, K. Boyer, C. K. Rhodes, J. C. Solem, R. S. Weinstein, “Design for a Fourier-transform holographic microscope,” in X-Ray Microscopy II, D. Sayre, M. Howells, J. Kirz, H. Rarback, eds. (Springer-Verlag, Berlin, 1988), pp. 284–287; W. S. Haddad, J. C. Solem, D. Cullen, K. Boyer, C. K. Rhodes, “A description of the theory and apparatus for digital reconstruction of Fourier transform holograms,” in Electronics Imaging ‘87, advance printing of paper summaries (Institute for Graphic Communication, Boston, Mass., 1987), Vol. II, pp. 683–688; W. S. Haddad, D. Cullen, J. C. Solem, K. Boyer, C. K. Rhodes, “X-ray Fourier-transform holographic microscope,” in Short Wavelength Coherent Radiation: Generation and Applications, R. W. Falcone, J. Kirz, eds., Vol. 2 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1988), pp. 284–289.

J. C. Solem, G. C. Baldwin, G. F. Chapline, “Holography at x-ray wavelengths,” in Proceedings of International Conference on Lasers 1981, C. B. Collins, ed. (STS, McClean, Va, 1981), pp. 296–235; J. C. Solem, G. C. Baldwin, “Microholography of living organisms,” Science 218, 229–235 (1982); J. C. Solem, G. F. Chapline, “X-ray biomicroholography,” Opt. Eng. 23, 193–203 (1984); I. McNulty, J. Kirz, C. Jacobsen, M. R. Howells, E. H. Anderson, “First results with a Fourier transform holographic microscope,” in X-Ray Microscopy III, A. G. Michette, G. R. Morrison, C. J. Buckley, eds. (Springer-Verlag, Berlin, 1992), 251–254.
[CrossRef] [PubMed]

Stroke, G.

G. Stroke, “Attainment of high resolutions in image-forming x-ray microscopy with ‘lensless’ Fourier-transform holograms and correlative source-effect compensation,” in Optique des Rayons X et Microanalyse (Hermann, Paris, 1966), pp. 30–46.

Upatnieks, J.

van der Voort, H. T. M.

H. T. M. van der Voort, G. J. Brakenhoff, “3-D image formation in high-aperture fluorescence confocal microscopy: a numerical analysis,” J. Microsc. (Oxford) 158, 43–54 (1989).
[CrossRef]

VanLigten, R.

R. VanLigten, H. Osterberg, “Holographic microscopy,” Nature 211, 282–283 (1966).
[CrossRef]

Weinstein, R. S.

W. S. Haddad, D. Cullen, K. Boyer, C. K. Rhodes, J. C. Solem, R. S. Weinstein, “Design for a Fourier-transform holographic microscope,” in X-Ray Microscopy II, D. Sayre, M. Howells, J. Kirz, H. Rarback, eds. (Springer-Verlag, Berlin, 1988), pp. 284–287; W. S. Haddad, J. C. Solem, D. Cullen, K. Boyer, C. K. Rhodes, “A description of the theory and apparatus for digital reconstruction of Fourier transform holograms,” in Electronics Imaging ‘87, advance printing of paper summaries (Institute for Graphic Communication, Boston, Mass., 1987), Vol. II, pp. 683–688; W. S. Haddad, D. Cullen, J. C. Solem, K. Boyer, C. K. Rhodes, “X-ray Fourier-transform holographic microscope,” in Short Wavelength Coherent Radiation: Generation and Applications, R. W. Falcone, J. Kirz, eds., Vol. 2 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1988), pp. 284–289.

White, H. E.

F. A. Jenkins, H. E. White, in Fundamentals of Optics, 4th ed. (McGraw-Hill, New York, 1976), pp. 71–72.

Winthrop, J.

J. Winthrop, C. Worthington, “X-ray microscopy by successive Fourier transformation,” Phys. Lett. 15, 124–126 (1965); G. Stroke, R. Restrick, “Holography with spatially noncoherent light,” Appl. Phys. Lett. 7, 229–230 (1966); G. Stroke, D. Falconer, “Attainment of high resolutions in wavefront reconstruction imaging—II,” J. Opt. Soc. Am. 55, 595 (1965).
[CrossRef]

Worthington, C.

J. Winthrop, C. Worthington, “X-ray microscopy by successive Fourier transformation,” Phys. Lett. 15, 124–126 (1965); G. Stroke, R. Restrick, “Holography with spatially noncoherent light,” Appl. Phys. Lett. 7, 229–230 (1966); G. Stroke, D. Falconer, “Attainment of high resolutions in wavefront reconstruction imaging—II,” J. Opt. Soc. Am. 55, 595 (1965).
[CrossRef]

J. Microsc. (Oxford)

H. T. M. van der Voort, G. J. Brakenhoff, “3-D image formation in high-aperture fluorescence confocal microscopy: a numerical analysis,” J. Microsc. (Oxford) 158, 43–54 (1989).
[CrossRef]

J. Opt. Soc. Am.

J. Opt. Soc. Am. A

Nature

D. Gabor, “A new microscopic principle,” Nature 161, 777–778 (1948); Proc. R. Soc. London Ser. A 197, 454–487 (1949).
[CrossRef] [PubMed]

R. VanLigten, H. Osterberg, “Holographic microscopy,” Nature 211, 282–283 (1966).
[CrossRef]

Phys. Lett.

J. Winthrop, C. Worthington, “X-ray microscopy by successive Fourier transformation,” Phys. Lett. 15, 124–126 (1965); G. Stroke, R. Restrick, “Holography with spatially noncoherent light,” Appl. Phys. Lett. 7, 229–230 (1966); G. Stroke, D. Falconer, “Attainment of high resolutions in wavefront reconstruction imaging—II,” J. Opt. Soc. Am. 55, 595 (1965).
[CrossRef]

Other

J. C. Solem, G. C. Baldwin, G. F. Chapline, “Holography at x-ray wavelengths,” in Proceedings of International Conference on Lasers 1981, C. B. Collins, ed. (STS, McClean, Va, 1981), pp. 296–235; J. C. Solem, G. C. Baldwin, “Microholography of living organisms,” Science 218, 229–235 (1982); J. C. Solem, G. F. Chapline, “X-ray biomicroholography,” Opt. Eng. 23, 193–203 (1984); I. McNulty, J. Kirz, C. Jacobsen, M. R. Howells, E. H. Anderson, “First results with a Fourier transform holographic microscope,” in X-Ray Microscopy III, A. G. Michette, G. R. Morrison, C. J. Buckley, eds. (Springer-Verlag, Berlin, 1992), 251–254.
[CrossRef] [PubMed]

W. S. Haddad, D. Cullen, K. Boyer, C. K. Rhodes, J. C. Solem, R. S. Weinstein, “Design for a Fourier-transform holographic microscope,” in X-Ray Microscopy II, D. Sayre, M. Howells, J. Kirz, H. Rarback, eds. (Springer-Verlag, Berlin, 1988), pp. 284–287; W. S. Haddad, J. C. Solem, D. Cullen, K. Boyer, C. K. Rhodes, “A description of the theory and apparatus for digital reconstruction of Fourier transform holograms,” in Electronics Imaging ‘87, advance printing of paper summaries (Institute for Graphic Communication, Boston, Mass., 1987), Vol. II, pp. 683–688; W. S. Haddad, D. Cullen, J. C. Solem, K. Boyer, C. K. Rhodes, “X-ray Fourier-transform holographic microscope,” in Short Wavelength Coherent Radiation: Generation and Applications, R. W. Falcone, J. Kirz, eds., Vol. 2 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1988), pp. 284–289.

F. A. Jenkins, H. E. White, in Fundamentals of Optics, 4th ed. (McGraw-Hill, New York, 1976), pp. 71–72.

G. Stroke, “Attainment of high resolutions in image-forming x-ray microscopy with ‘lensless’ Fourier-transform holograms and correlative source-effect compensation,” in Optique des Rayons X et Microanalyse (Hermann, Paris, 1966), pp. 30–46.

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Figures (5)

Fig. 1
Fig. 1

Schematic of a Fourier-transform microholographic camera configured for operation in the visible range; f.l., focal length.

Fig. 2
Fig. 2

Side-view schematic of the target system prepared for visible microholography.

Fig. 3
Fig. 3

Transverse PSF measured by imaging the edge of a microscope test reticle. The curve comprises 250 data points reconstructed with a transverse spacing of 10 nm.

Fig. 4
Fig. 4

Reconstructed image of an Ascaris in maturation stage. The cuticle and individual cells are visible. The cell diameter is ~ 40 μm. The nuclear region visible within some cells is ~ 20 μm in diameter.

Fig. 5
Fig. 5

Local region of Ascaris image involving 106 resolution elements (102 × 102 × 102). The region is centered around one cell that is adjacent to the cuticle. Features arising from the depth resolution are visible as the focal plane is positioned at different depths (a)–(d). See text for discussion.

Equations (3)

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ϕ ( x , y ) = exp ( i π x 2 + y 2 λ f a ) ,
f a = b c b - c ,
δ = 1.22 λ 2 n sin θ ,

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