Abstract

We present a general imaging technique called graded-field microscopy for obtaining phase-gradient contrast in biological tissue slices. The technique is based on introducing partial beam blocks in the illumination and detection apertures of a standard white-light widefield transillumination microscope. Depending on the relative aperture sizes, one block produces phase-gradient contrast while the other reduces brightfield background, allowing a full operating range between brightfield and darkfield contrast. We demonstrate graded-field imaging of neurons in a rat brain slice.

© 2006 Optical Society of America

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  1. F. Zernike, "Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung [in German],"Z. Tech. Phys. 16,454 (1935).
  2. F. Zernike, "How I discovered phase contrast,"Science 121,345-349 (1955).
    [CrossRef] [PubMed]
  3. G. Nomarski, "Microinterf´erom`etre diff´erentiel `a ondes polaris´ees [in French]," J. Phys. Radium 16,S9 (1955).
  4. R. D. Allen, G. B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
    [PubMed]
  5. B. Kachar, "Asymmetric illumination contrast: a method of image formation for video microscopy,"Science 227,766-768 (1985).
    [CrossRef] [PubMed]
  6. W. B. Piekos, "Diffracted-light contrast enhancement: A re-examination of oblique illumination," Micros. Res. Tech. 46,334-337 (1999).
    [CrossRef]
  7. S. Inoue, Video Microscopy (Plenum Press, New York, 1986).
  8. H. U. Dodt, M. Eder, A. Frick, W. Zieglg¨ansberger, "Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation", Science 286,111-113 (1999).
    [CrossRef]
  9. C. F. Saylor, "Accuracy of microscopical methods for determining refractive index by immersion,"J. Res. US Natl. Bur. Stds. 15,277 (1935).
  10. E. H. Linfoot, Recent advances in optics (Clarendon Press, Oxford, 1955).
  11. J. Ojeda-Castaneda, L. R. Berriel-Valdos, "Classification scheme and properties of schlieren techniques," Appl. Opt. 16,18, 3338-3341 (1979).
    [CrossRef]
  12. J. G. Dodd, "Interferometry with Schlieren microscopy," Appl. Opt. 16,16, 470-472 (1977).
    [CrossRef] [PubMed]
  13. D. Axelrod, "Zero-cost modification of bright field microscopes for imaging phase gradient on cells: Schlieren optics,"Cell Biophys. 3,167-173 (1981).
    [PubMed]
  14. S. Lowenthal, Y. Belvaux, "Observation of phase objects by optically processed Hilbert transform,"Appl. Phys. Lett. 11, 49-51 (1967).
    [CrossRef]
  15. R. Hoffman and L. Gross, "Modulation contrast microscopy," Appl. Opt. 14,1169-1176 (1975).
    [CrossRef] [PubMed]
  16. M. Born and E. Wolf, Principles of optics (Cambridge University Press, Cambridge, UK, 1999).

1999

W. B. Piekos, "Diffracted-light contrast enhancement: A re-examination of oblique illumination," Micros. Res. Tech. 46,334-337 (1999).
[CrossRef]

H. U. Dodt, M. Eder, A. Frick, W. Zieglg¨ansberger, "Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation", Science 286,111-113 (1999).
[CrossRef]

1985

B. Kachar, "Asymmetric illumination contrast: a method of image formation for video microscopy,"Science 227,766-768 (1985).
[CrossRef] [PubMed]

1981

D. Axelrod, "Zero-cost modification of bright field microscopes for imaging phase gradient on cells: Schlieren optics,"Cell Biophys. 3,167-173 (1981).
[PubMed]

1979

J. Ojeda-Castaneda, L. R. Berriel-Valdos, "Classification scheme and properties of schlieren techniques," Appl. Opt. 16,18, 3338-3341 (1979).
[CrossRef]

1977

1975

1969

R. D. Allen, G. B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

1967

S. Lowenthal, Y. Belvaux, "Observation of phase objects by optically processed Hilbert transform,"Appl. Phys. Lett. 11, 49-51 (1967).
[CrossRef]

1955

F. Zernike, "How I discovered phase contrast,"Science 121,345-349 (1955).
[CrossRef] [PubMed]

G. Nomarski, "Microinterf´erom`etre diff´erentiel `a ondes polaris´ees [in French]," J. Phys. Radium 16,S9 (1955).

1935

F. Zernike, "Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung [in German],"Z. Tech. Phys. 16,454 (1935).

C. F. Saylor, "Accuracy of microscopical methods for determining refractive index by immersion,"J. Res. US Natl. Bur. Stds. 15,277 (1935).

Allen, R. D.

R. D. Allen, G. B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

Axelrod, D.

D. Axelrod, "Zero-cost modification of bright field microscopes for imaging phase gradient on cells: Schlieren optics,"Cell Biophys. 3,167-173 (1981).
[PubMed]

Belvaux, Y.

S. Lowenthal, Y. Belvaux, "Observation of phase objects by optically processed Hilbert transform,"Appl. Phys. Lett. 11, 49-51 (1967).
[CrossRef]

Berriel-Valdos, L. R.

J. Ojeda-Castaneda, L. R. Berriel-Valdos, "Classification scheme and properties of schlieren techniques," Appl. Opt. 16,18, 3338-3341 (1979).
[CrossRef]

David, G. B.

R. D. Allen, G. B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

Dodd, J. G.

Dodt, H. U.

H. U. Dodt, M. Eder, A. Frick, W. Zieglg¨ansberger, "Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation", Science 286,111-113 (1999).
[CrossRef]

Eder, M.

H. U. Dodt, M. Eder, A. Frick, W. Zieglg¨ansberger, "Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation", Science 286,111-113 (1999).
[CrossRef]

Frick, A.

H. U. Dodt, M. Eder, A. Frick, W. Zieglg¨ansberger, "Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation", Science 286,111-113 (1999).
[CrossRef]

Gross, L.

Hoffman, R.

Kachar, B.

B. Kachar, "Asymmetric illumination contrast: a method of image formation for video microscopy,"Science 227,766-768 (1985).
[CrossRef] [PubMed]

Lowenthal, S.

S. Lowenthal, Y. Belvaux, "Observation of phase objects by optically processed Hilbert transform,"Appl. Phys. Lett. 11, 49-51 (1967).
[CrossRef]

Nomarski, G.

R. D. Allen, G. B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

G. Nomarski, "Microinterf´erom`etre diff´erentiel `a ondes polaris´ees [in French]," J. Phys. Radium 16,S9 (1955).

Ojeda-Castaneda, J.

J. Ojeda-Castaneda, L. R. Berriel-Valdos, "Classification scheme and properties of schlieren techniques," Appl. Opt. 16,18, 3338-3341 (1979).
[CrossRef]

Piekos, W. B.

W. B. Piekos, "Diffracted-light contrast enhancement: A re-examination of oblique illumination," Micros. Res. Tech. 46,334-337 (1999).
[CrossRef]

Saylor, C. F.

C. F. Saylor, "Accuracy of microscopical methods for determining refractive index by immersion,"J. Res. US Natl. Bur. Stds. 15,277 (1935).

Zernike, F.

F. Zernike, "How I discovered phase contrast,"Science 121,345-349 (1955).
[CrossRef] [PubMed]

F. Zernike, "Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung [in German],"Z. Tech. Phys. 16,454 (1935).

Appl. Opt.

Appl. Phys. Lett.

S. Lowenthal, Y. Belvaux, "Observation of phase objects by optically processed Hilbert transform,"Appl. Phys. Lett. 11, 49-51 (1967).
[CrossRef]

Cell Biophys.

D. Axelrod, "Zero-cost modification of bright field microscopes for imaging phase gradient on cells: Schlieren optics,"Cell Biophys. 3,167-173 (1981).
[PubMed]

J. Phys. Radium

G. Nomarski, "Microinterf´erom`etre diff´erentiel `a ondes polaris´ees [in French]," J. Phys. Radium 16,S9 (1955).

J. Res. US Natl. Bur. Stds.

C. F. Saylor, "Accuracy of microscopical methods for determining refractive index by immersion,"J. Res. US Natl. Bur. Stds. 15,277 (1935).

Micros. Res. Tech.

W. B. Piekos, "Diffracted-light contrast enhancement: A re-examination of oblique illumination," Micros. Res. Tech. 46,334-337 (1999).
[CrossRef]

Science

B. Kachar, "Asymmetric illumination contrast: a method of image formation for video microscopy,"Science 227,766-768 (1985).
[CrossRef] [PubMed]

H. U. Dodt, M. Eder, A. Frick, W. Zieglg¨ansberger, "Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation", Science 286,111-113 (1999).
[CrossRef]

F. Zernike, "How I discovered phase contrast,"Science 121,345-349 (1955).
[CrossRef] [PubMed]

Z. Tech. Phys.

F. Zernike, "Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung [in German],"Z. Tech. Phys. 16,454 (1935).

Z. Wiss. Mikrosk.

R. D. Allen, G. B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
[PubMed]

Other

S. Inoue, Video Microscopy (Plenum Press, New York, 1986).

E. H. Linfoot, Recent advances in optics (Clarendon Press, Oxford, 1955).

M. Born and E. Wolf, Principles of optics (Cambridge University Press, Cambridge, UK, 1999).

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Figures (3)

Fig. 1.
Fig. 1.

Experimental setup: incoherent white light transilluminates a sample in a 6 f (unit-magnification) imaging line. Partial beam blocks are introduced in the illumination (top) and/or detection (bottom) apertures.

Fig. 2.
Fig. 2.

Plots of the window functions Ke(0, x1d) and Ko (0, x 1d ) for different aperture block configurations (shown schematically on left). Ke reveals amplitude fluctuations whereas Ko reveals phase-gradient fluctuations in the sample. B characterizes the net brightfield contribution, or background level, as defined by the integral of Ke (Eq. (26)). The argument x 1d is normalized to be unitless.

Fig. 3.
Fig. 3.

Experimental images of pyramidal neurons in an acute rat hippocampus slice (depth ~50µm). Images were taken with no aperture blocks (fully brightfield configuration - panel A), with a partial beam block in the illumination (panel B) or (panel C) detection aperture, and with partial beam blocks in both apertures (panel D). In the experimental setup, the illumination aperture was larger than the detection aperture. The slices were 400mm thick; the rat was 15 days old.

Equations (33)

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E 1 ( x 1 ) = α 1 α 2 E 0 ( ξ 0 ) e i x 1 ξ 0 d ξ 0
J ( x , x ) = E ( x ) E * ( x ) ¯
J 1 in ( x 1 , x 1 ) = α 1 α 2 d ξ 0 α 1 α 2 d ξ 0 J 0 ( ξ 0 , ξ 0 ) e i ( x 1 ξ 0 x 1 ξ 0 )
J 0 ( ξ 0 , ξ 0 ) = δ ( ξ 0 ξ 0 )
J 1 in ( x 1 , x 1 ) = J 1 in ( x 1 d ) = α d e i α c x 1 d sinc ( 1 2 α d x 1 d )
α d = α 2 α 1
α c = 1 2 ( α 1 + α 2 )
J 3 ( x 3 , x 3 ) = β 1 β 2 d ξ 2 β 1 β 2 d ξ 2 J 2 ( ξ 2 , ξ 2 ) e i ( x 3 ξ 2 x 3 ξ 2 )
J 2 ( ξ 2 , ξ 2 ) = d x 1 d x 1 J 1 out ( x 1 , x 1 ) e i ( ξ 2 x 1 ξ 2 x 1 )
J 3 ( x 3 , x 3 ) = β d 2 d x 1 d x 1 J 1 out ( x 1 , x 1 ) e i β c ( x 1 d + x 3 d ) sinc ( 1 2 β d ( x 1 + x 3 ) ) sinc ( 1 2 β d ( x 1 + x 3 ) )
I 3 ( x 3 ) = d x 1 c d x 1 d G 13 ( x 3 + x 1 c , x 1 d ) J 1 out ( x 1 c , x 1 d )
G 13 ( x 3 + x 1 c , x 1 d ) = β d 2 e i β c x 1 d sinc ( 1 2 β d ( x 3 + x 1 c 1 2 x 1 d ) ) sinc ( 1 2 β d ( x 3 + x 1 c + 1 2 x 1 d ) )
E 1 out ( x 1 ) = t ( x 1 ) E 1 in ( x 1 )
J 1 out ( x 1 c , x 1 d ) = T ( x 1 c , x 1 d ) J 1 in ( x 1 c , x 1 d )
T ( x 1 c , x 1 d ) = t ( x 1 c 1 2 x 1 d ) t * ( x 1 c + 1 2 x 1 d )
T ( x 1 c , x 1 d ) = T ( x 1 c x 1 d ) *
J r ( x 1 d ) = α d cos ( α c x 1 d ) sin c ( 1 2 α d x 1 d )
J i ( x 1 d ) = α d sin ( α c x 1 d ) sinc ( 1 2 α d x 1 d )
G r ( x 3 + x 1 c , x 1 d ) = β d 2 cos ( β c x 1 d ) sinc ( 1 2 β d ( x 3 + x 1 c 1 2 x 1 d ) ) sinc ( 1 2 β d ( x 3 + x 1 c + 1 2 x 1 d ) )
G i ( x 3 + x 1 c , x 1 d ) = β d 2 sin ( β c x 1 d ) sinc ( 1 2 β d ( x 3 + x 1 c 1 2 x 1 d ) ) sinc ( 1 2 β d ( x 3 + x 1 c + 1 2 x 1 d ) )
K e ( x 3 + x 1 c , x 1 d ) = G r ( x 3 + x 1 c , x 1 d ) J r ( x 1 d ) G i ( x 3 + x 1 c , x 1 d ) J i ( x 1 d )
K o ( x 3 + x 1 c , x 1 d ) = G i ( x 3 + x 1 c , x 1 d ) J r ( x 1 d ) + G r ( x 3 + x 1 c , x 1 d ) J i ( x 1 d )
K e ( x 3 + x 1 c , x 1 d ) = cos ( ( α c + β c ) x 1 d ) K ( x 3 + x 1 c , x 1 d )
K o ( x 3 + x 1 c , x 1 d ) = sin ( ( α c + β c ) x 1 d ) K ( x 3 + x 1 c , x 1 d )
K ( x 3 + x 1 c , x 1 d ) = α d β d 2 sinc ( 1 2 α d x 1 d ) sinc ( 1 2 β d ( x 3 + x 1 c 1 2 x 1 d ) ) sinc ( 1 2 β d ( x 3 + x 1 c + 1 2 x 1 d ) )
I 3 ( x 3 ) = ( K e ( x 3 + x 1 c , x 1 d ) T r ( x 1 c , x 1 d ) + K o ( x 3 + x 1 c , x 1 d ) T i ( x 1 c , x 1 d ) ) d x 1 c d x 1 d
B = K e ( x 3 + x 1 c , x 1 d ) d x 1 c d x 1 d K total ( x 3 + x 1 c , x 1 c ) d x 1 c d x 1 d
sinc ( 1 2 β d ( x 3 + x 1 c + 1 2 x 1 d ) ) sinc ( 1 2 β d ( x 3 + x 1 c 1 2 x 1 d ) ) d x 1 c = 2 π β d sinc ( 1 2 β d x 1 d )
t ( x 1 ) = 1 q ( x 1 ) + ip ( x 1 )
T r ( x 1 c , x 1 d ) 1 q ( x 1 c 1 2 x 1 d ) q ( x 1 c + 1 2 x 1 d )
T i ( x 1 c , x 1 d ) p ( x 1 c + 1 2 x 1 d ) p ( x 1 c 1 2 x 1 d )
K ( x 3 + x 1 c , x 1 d ) α d β d 2 sinc ( 1 2 α d x 1 d ) sinc 2 ( 1 2 β d ( x 3 + x 1 c ) )
K ( x 3 + x 1 c , x 1 d ) α d β d 2 sinc ( 1 2 β d ( x 3 + x 1 c 1 2 x 1 d ) ) sinc ( 1 2 β d ( x 3 + x 1 c + 1 2 x 1 d ) )

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