Abstract

Autoconfocal microscopy (ACM) is a simple implementation of a transmitted-light confocal microscopy where a nonlinear detector plays the role of a virtual self-aligned pinhole. We report here a significant improvement of ACM based on the use of graded-field (GF) imaging. The technique of GF imaging involves introducing partial beam blocks in the illumination and detection apertures of an imaging system. These partial beam blocks confer phase-gradient sensitivity to the imaging system and allow control over its background level. We present the theory of the GF contrast in the context of ACM, comparing it to GF contrast in a non-scanning widefield microscope, and discuss various performance characteristics of GF-ACM in terms of resolution, sectioning strength, and an “under-detection” light collection geometry. An advantage of ACM is that it can be readily combined with two-photon excited fluorescence (TPEF) microscopy. We present images of rat brain hippocampus using simultaneous GF-ACMand TPEF microscopy. These images are inherently co-registered.

© 2007 Optical Society of America

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References

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  1. A.E. Dixon and C. Cogswell, "Confocal microscopy with transmitted light," in Handbook of Biological Confocal Microscopy, J.B. Pawley, ed. (Plenum Press, 1995), pp. 479-490.
  2. C. Yang and J. Mertz, "Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection," Opt. Lett. 28,224-227 (2003).
  3. T. Pons and J. Mertz, "Autoconfocal microscopy with nonlinear transmitted light detection," J. Opt. Soc. Am. B 21,1486-1493 (2004).
    [CrossRef]
  4. R. Yi, K.K. Chu, and J. Mertz, "Graded-field microscopy with white light," Opt. Express 14,5191-5200 (2006).
    [CrossRef]
  5. G. Nomarski, "Microinterferometre differentiel a ondes polarisees [in French]," J. Phys. Radium 16,S9 (1955).
  6. R.D. Allen, G.B. David, G. Nomarski, "The Zeiss-Nomarski differential interference equipment for transmittedlight microscopy," Z. Wiss. Mikrosk. 69,193-221 (1969).
  7. W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
    [CrossRef]
  8. Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
    [CrossRef]
  9. T. Wilson, "The role of the pinhole in confocal imaging system," in Handbook of Biological Confocal Microscopy, J.B. Pawley, ed. (Plenum Press, 1995), pp. 167-182.
  10. N. Streibl, "Depth transfer by an imaging system," Opt. Acta 31,1233-1241 (1984).
  11. M. Born and E. Wolf, Principles of optics (Cambridge University Press, Cambridge, UK, 1999).

2006 (1)

2004 (1)

2003 (2)

C. Yang and J. Mertz, "Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection," Opt. Lett. 28,224-227 (2003).

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef]

1999 (1)

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef]

1984 (1)

N. Streibl, "Depth transfer by an imaging system," Opt. Acta 31,1233-1241 (1984).

1969 (1)

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

1955 (1)

G. Nomarski, "Microinterferometre differentiel a ondes polarisees [in French]," J. Phys. Radium 16,S9 (1955).

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).

Amos, W.B.

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef]

Cattermole, D.M.

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef]

Chu, K.K.

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).

Hayashi, Y.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef]

Laufer, J.

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef]

Mainen, Z.F.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef]

Maletic-Savatic, M.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef]

Malinow, R.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef]

Mertz, J.

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).

G. Nomarski, "Microinterferometre differentiel a ondes polarisees [in French]," J. Phys. Radium 16,S9 (1955).

Pons, T.

Reichelt, S.

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef]

Shi, S.H.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef]

Streibl, N.

N. Streibl, "Depth transfer by an imaging system," Opt. Acta 31,1233-1241 (1984).

Svoboda, K.

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef]

Yang, C.

Yi, R.

J. Microsc. (1)

W.B. Amos, S. Reichelt, D.M. Cattermole, and J. Laufer, "Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics," J. Microsc. 210,166-175 (2003).
[CrossRef]

J. Opt. Soc. Am. B (1)

J. Phys. Radium (1)

G. Nomarski, "Microinterferometre differentiel a ondes polarisees [in French]," J. Phys. Radium 16,S9 (1955).

Methods (1)

Z.F. Mainen, M. Maletic-Savatic, S.H. Shi, Y. Hayashi, R. Malinow, K. Svoboda, "Two-photon imaging in living brain slices," Methods 18,231-239 (1999).
[CrossRef]

Opt. Acta (1)

N. Streibl, "Depth transfer by an imaging system," Opt. Acta 31,1233-1241 (1984).

Opt. Express (1)

Opt. Lett. (1)

Z. Wiss. Mikrosk. (1)

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

Other (3)

T. Wilson, "The role of the pinhole in confocal imaging system," in Handbook of Biological Confocal Microscopy, J.B. Pawley, ed. (Plenum Press, 1995), pp. 167-182.

A.E. Dixon and C. Cogswell, "Confocal microscopy with transmitted light," in Handbook of Biological Confocal Microscopy, J.B. Pawley, ed. (Plenum Press, 1995), pp. 479-490.

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

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