Abstract

Ultramicroscopy allows for the 3D reconstruction of centimeter sized samples with a spatial resolution of several micrometers. Nevertheless, in poorly cleared or very large specimens the images may suffer from blurring and low contrast levels. To address these problems, ultramicroscopy was combined with the principle of confocal microscopy using a slowly rotating Nipkow disk. This configuration was tested by comparing images from mouse hippocampal neurons and mouse liver blood vessels recorded in confocal and conventional mode. It was found that confocality minimizes the background noise and considerably improves the signal-to-noise ratio when applied to ultramicroscopy.

© 2009 Optical Society of America

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  1. C. J. R. Sheppard and R. Kompfner, Appl. Opt. 17, 2879 (1978).
    [CrossRef] [PubMed]
  2. W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  3. S. W. Hell, Nat. Biotechnol. 21, 1347 (2003).
    [CrossRef] [PubMed]
  4. H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
    [CrossRef] [PubMed]
  5. K. Becker, N. Jährling, E. R. Kramer, F. Schnorrer, and H.-U. Dodt, J. Biophoton 1, 36 (2008).
    [CrossRef]
  6. W. Spalteholz, Über das Durchsichtigmachen von menschlichen und tierischen Präparaten (S. Hirzel, Leipzig, 1914).
  7. W. Lukosz and M. Marchand, J. Mod. Opt. 10, 241 (1963).
  8. M. A. A. Neil, R. Juskaitis, and T. Wilson, Opt. Lett. 22, 1905 (1997).
    [CrossRef]
  9. V. Poher, H. X. Zhang, G. T. Kennedy, C. Griffin, S. Oddos, E. Gu, D. S. Elson, J. M. Girkin, P. M. W. French, M. D. Dawson, and M. A. A. Neil, Opt. Express 15, 11196 (2007).
    [CrossRef] [PubMed]
  10. T. Breuninger, K. Greger, and E. H. K. Stelzer, Opt. Lett. 32, 1938 (2007).
    [CrossRef] [PubMed]
  11. M. G. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 102, 13081 (2005).
    [CrossRef] [PubMed]
  12. P. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, U.S. patent 6,016,367, 18 January 2000.
  13. R. Heintzmann and P. Benedetti, Appl. Opt. 45, 5037 (2006).
    [CrossRef] [PubMed]
  14. H.-U. Dodt and K. Becker, Proc. SPIE 5139, 79 (2003).
    [CrossRef]
  15. J. B. Pawley, The Handbook of Biological Confocal Microscopy, 3rd ed. (Plenum, 2006), p. 268.

2008 (1)

K. Becker, N. Jährling, E. R. Kramer, F. Schnorrer, and H.-U. Dodt, J. Biophoton 1, 36 (2008).
[CrossRef]

2007 (3)

2006 (1)

2005 (1)

M. G. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 102, 13081 (2005).
[CrossRef] [PubMed]

2003 (2)

S. W. Hell, Nat. Biotechnol. 21, 1347 (2003).
[CrossRef] [PubMed]

H.-U. Dodt and K. Becker, Proc. SPIE 5139, 79 (2003).
[CrossRef]

1997 (1)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

1978 (1)

1963 (1)

W. Lukosz and M. Marchand, J. Mod. Opt. 10, 241 (1963).

Becker, K.

K. Becker, N. Jährling, E. R. Kramer, F. Schnorrer, and H.-U. Dodt, J. Biophoton 1, 36 (2008).
[CrossRef]

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

H.-U. Dodt and K. Becker, Proc. SPIE 5139, 79 (2003).
[CrossRef]

Benedetti, P.

R. Heintzmann and P. Benedetti, Appl. Opt. 45, 5037 (2006).
[CrossRef] [PubMed]

P. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, U.S. patent 6,016,367, 18 January 2000.

Breuninger, T.

Dawson, M. D.

Deininger, K.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Deussing, J. M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Dodt, H.-U.

K. Becker, N. Jährling, E. R. Kramer, F. Schnorrer, and H.-U. Dodt, J. Biophoton 1, 36 (2008).
[CrossRef]

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

H.-U. Dodt and K. Becker, Proc. SPIE 5139, 79 (2003).
[CrossRef]

Eder, M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Elson, D. S.

Evangelista, V.

P. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, U.S. patent 6,016,367, 18 January 2000.

French, P. M. W.

Girkin, J. M.

Greger, K.

Griffin, C.

Gu, E.

Guidarini, D.

P. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, U.S. patent 6,016,367, 18 January 2000.

Gustafsson, M. G.

M. G. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 102, 13081 (2005).
[CrossRef] [PubMed]

Heintzmann, R.

Hell, S. W.

S. W. Hell, Nat. Biotechnol. 21, 1347 (2003).
[CrossRef] [PubMed]

Jährling, N.

K. Becker, N. Jährling, E. R. Kramer, F. Schnorrer, and H.-U. Dodt, J. Biophoton 1, 36 (2008).
[CrossRef]

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Juskaitis, R.

Kennedy, G. T.

Kompfner, R.

Kramer, E. R.

K. Becker, N. Jährling, E. R. Kramer, F. Schnorrer, and H.-U. Dodt, J. Biophoton 1, 36 (2008).
[CrossRef]

Leischner, U.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Lukosz, W.

W. Lukosz and M. Marchand, J. Mod. Opt. 10, 241 (1963).

Marchand, M.

W. Lukosz and M. Marchand, J. Mod. Opt. 10, 241 (1963).

Mauch, C. P.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Neil, M. A. A.

Oddos, S.

Pawley, J. B.

J. B. Pawley, The Handbook of Biological Confocal Microscopy, 3rd ed. (Plenum, 2006), p. 268.

Poher, V.

Schierloh, A.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Schnorrer, F.

K. Becker, N. Jährling, E. R. Kramer, F. Schnorrer, and H.-U. Dodt, J. Biophoton 1, 36 (2008).
[CrossRef]

Sheppard, C. J. R.

Spalteholz, W.

W. Spalteholz, Über das Durchsichtigmachen von menschlichen und tierischen Präparaten (S. Hirzel, Leipzig, 1914).

Stelzer, E. H. K.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Vestri, S.

P. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, U.S. patent 6,016,367, 18 January 2000.

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Wilson, T.

Zhang, H. X.

Zieglgänsberger, W.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Appl. Opt. (2)

J. Biophoton (1)

K. Becker, N. Jährling, E. R. Kramer, F. Schnorrer, and H.-U. Dodt, J. Biophoton 1, 36 (2008).
[CrossRef]

J. Mod. Opt. (1)

W. Lukosz and M. Marchand, J. Mod. Opt. 10, 241 (1963).

Nat. Biotechnol. (1)

S. W. Hell, Nat. Biotechnol. 21, 1347 (2003).
[CrossRef] [PubMed]

Nat. Methods (1)

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Methods 4, 331 (2007).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (2)

Proc. Natl. Acad. Sci. U.S.A. (1)

M. G. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 102, 13081 (2005).
[CrossRef] [PubMed]

Proc. SPIE (1)

H.-U. Dodt and K. Becker, Proc. SPIE 5139, 79 (2003).
[CrossRef]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Other (3)

W. Spalteholz, Über das Durchsichtigmachen von menschlichen und tierischen Präparaten (S. Hirzel, Leipzig, 1914).

J. B. Pawley, The Handbook of Biological Confocal Microscopy, 3rd ed. (Plenum, 2006), p. 268.

P. Benedetti, V. Evangelista, D. Guidarini, and S. Vestri, U.S. patent 6,016,367, 18 January 2000.

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Figures (4)

Fig. 1
Fig. 1

Optical setup of the confocal ultramicroscope. The laser beam is expanded by a 10 × beam expander (BE), passed through an aperture (A), and focused by a cylindrical lens (CL, f = 80 mm ) onto a slowly rotating Nipkow disk (ND). The spatially modulated light sheet is then projected into the specimen 1:1 by two identical plano-convex lenses (PCL).

Fig. 2
Fig. 2

Comparison of conventional and confocal ultramicroscopy. (a) Conventional UM and (b) confocal UM image of a mouse hippocampus. The frame marks the area of the detail images (c) and (d). The white arrow in (c), (d) points to a dendrite of a CA1-pyramidal neuron that was revealed. Objective: Olympus 10 × , NA 0.30, γ M = 0.5 .

Fig. 3
Fig. 3

Contrast enhancement analysis by comparison of intensity profiles of a conventional UM image (dotted–dashed curve) and a confocal UM image (solid curve) of the mouse hippocampus. The white line in the inset marks the cut through the images, along which the intensity profile was measured.

Fig. 4
Fig. 4

Blood vessels in an autofluorescent mouse liver. Comparison of (a) a conventional UM image, (b) a single confocal UM image, and (c) the processed image composed of 40 single images. Objective: Olympus 4 × , NA 0.28, γ M = 0.5 .

Equations (1)

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I rec ( x , y ) = Max n = 0 N I n ( x , y ) γ M Min n = 0 N I n ( x , y ) .

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