Abstract

The macromolecular structure of purified cellulose samples is studied by second-harmonic generation (SHG) imaging microscopy. We show that the SHG contrast in both Valonia and Acetobacter cellulose strongly resembles that of collagen from animal tissues, both in terms of morphology and polarization anisotropy. Polarization analysis shows that microfibrils in each lamella are highly aligned and ordered and change directions by 90° in adjacent lamellae. The angular dependence of the SHG intensity fits well to a cos2 θ distribution, which is characteristic of the electric dipole interaction. Enzymatic degradation of Valonia fibers by cellulase is followed in real time by SHG imaging and results in exponential decay kinetics, showing that SHG imaging microscopy is ideal for monitoring dynamics in biological systems.

© 2003 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef] [PubMed]
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2002 (2)

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 82, 493 (2002).
[CrossRef]

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. U.S.A. 99, 11014 (2002).
[CrossRef]

2001 (2)

V. Ottani, M. Raspanti, and A. Ruggeri, Micron 32, 251 (2001).
[CrossRef]

T. Kondo, E. Togawa, and J. R. M. Brown, Biomacromolecules 2, 1324 (2001).
[CrossRef]

2000 (1)

P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (2000).
[CrossRef]

1998 (2)

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

K. Beck and B. Brodsky, J. Struct. Biol. 122, 17 (1998).
[CrossRef]

1997 (1)

S. K. Cousins and J. R. M. Brown, Polymer 38, 897 (1997).
[CrossRef]

1996 (2)

J. R. M. Brown, Pure Appl. Chem. 10, 1345 (1996).

K. Kudlicka, J. H. Lee, and J. R. M. Brown, Am. J. Bot. 83, 274 (1996).
[CrossRef]

1994 (1)

J. D. Beyers, H. I. Lee, T. Petralli-Mallow, and J. M. Hicks, Phys. Rev. B 49, 1464 (1994).

1986 (1)

S. Inoue, Video Microscopy (Plenum, New York, 1986).
[CrossRef]

Beck, K.

K. Beck and B. Brodsky, J. Struct. Biol. 122, 17 (1998).
[CrossRef]

Beyers, J. D.

J. D. Beyers, H. I. Lee, T. Petralli-Mallow, and J. M. Hicks, Phys. Rev. B 49, 1464 (1994).

Brodsky, B.

K. Beck and B. Brodsky, J. Struct. Biol. 122, 17 (1998).
[CrossRef]

Brown, J. R. M.

T. Kondo, E. Togawa, and J. R. M. Brown, Biomacromolecules 2, 1324 (2001).
[CrossRef]

S. K. Cousins and J. R. M. Brown, Polymer 38, 897 (1997).
[CrossRef]

J. R. M. Brown, Pure Appl. Chem. 10, 1345 (1996).

K. Kudlicka, J. H. Lee, and J. R. M. Brown, Am. J. Bot. 83, 274 (1996).
[CrossRef]

Campagnola, P. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 82, 493 (2002).
[CrossRef]

P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (2000).
[CrossRef]

Cousins, S. K.

S. K. Cousins and J. R. M. Brown, Polymer 38, 897 (1997).
[CrossRef]

Elshocht, S. V.

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

Hellemans, L.

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

Hicks, J. M.

J. D. Beyers, H. I. Lee, T. Petralli-Mallow, and J. M. Hicks, Phys. Rev. B 49, 1464 (1994).

Hoppe, P. E.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 82, 493 (2002).
[CrossRef]

Inoue, S.

S. Inoue, Video Microscopy (Plenum, New York, 1986).
[CrossRef]

Katz, T. J.

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

Kauranen, M.

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

Kondo, T.

T. Kondo, E. Togawa, and J. R. M. Brown, Biomacromolecules 2, 1324 (2001).
[CrossRef]

Kudlicka, K.

K. Kudlicka, J. H. Lee, and J. R. M. Brown, Am. J. Bot. 83, 274 (1996).
[CrossRef]

Lee, H. I.

J. D. Beyers, H. I. Lee, T. Petralli-Mallow, and J. M. Hicks, Phys. Rev. B 49, 1464 (1994).

Lee, J. H.

K. Kudlicka, J. H. Lee, and J. R. M. Brown, Am. J. Bot. 83, 274 (1996).
[CrossRef]

Lewis, A.

P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (2000).
[CrossRef]

Loew, L. M.

P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (2000).
[CrossRef]

Malone, C. J.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 82, 493 (2002).
[CrossRef]

Millard, A. C.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 82, 493 (2002).
[CrossRef]

Mohler, W. A.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 82, 493 (2002).
[CrossRef]

Nuckolls, C.

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

Ottani, V.

V. Ottani, M. Raspanti, and A. Ruggeri, Micron 32, 251 (2001).
[CrossRef]

Persoons, A.

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

Petralli-Mallow, T.

J. D. Beyers, H. I. Lee, T. Petralli-Mallow, and J. M. Hicks, Phys. Rev. B 49, 1464 (1994).

Raspanti, M.

V. Ottani, M. Raspanti, and A. Ruggeri, Micron 32, 251 (2001).
[CrossRef]

Ruggeri, A.

V. Ottani, M. Raspanti, and A. Ruggeri, Micron 32, 251 (2001).
[CrossRef]

Snauwaert, J.

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

Terasaki, M.

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 82, 493 (2002).
[CrossRef]

Togawa, E.

T. Kondo, E. Togawa, and J. R. M. Brown, Biomacromolecules 2, 1324 (2001).
[CrossRef]

Tromberg, B. J.

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. U.S.A. 99, 11014 (2002).
[CrossRef]

Verbiest, T.

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

Wei, M. D.

P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (2000).
[CrossRef]

Yeh, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. U.S.A. 99, 11014 (2002).
[CrossRef]

Zoumi, A.

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. U.S.A. 99, 11014 (2002).
[CrossRef]

Am. J. Bot. (1)

K. Kudlicka, J. H. Lee, and J. R. M. Brown, Am. J. Bot. 83, 274 (1996).
[CrossRef]

Biomacromolecules (1)

T. Kondo, E. Togawa, and J. R. M. Brown, Biomacromolecules 2, 1324 (2001).
[CrossRef]

Biophys. J. (2)

P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, Biophys. J. 82, 493 (2002).
[CrossRef]

P. J. Campagnola, M. D. Wei, A. Lewis, and L. M. Loew, Biophys. J. 77, 3341 (2000).
[CrossRef]

J. Struct. Biol. (1)

K. Beck and B. Brodsky, J. Struct. Biol. 122, 17 (1998).
[CrossRef]

Micron (1)

V. Ottani, M. Raspanti, and A. Ruggeri, Micron 32, 251 (2001).
[CrossRef]

Phys. Rev. B (1)

J. D. Beyers, H. I. Lee, T. Petralli-Mallow, and J. M. Hicks, Phys. Rev. B 49, 1464 (1994).

Polymer (1)

S. K. Cousins and J. R. M. Brown, Polymer 38, 897 (1997).
[CrossRef]

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

A. Zoumi, A. Yeh, and B. J. Tromberg, Proc. Natl. Acad. Sci. U.S.A. 99, 11014 (2002).
[CrossRef]

Pure Appl. Chem. (1)

J. R. M. Brown, Pure Appl. Chem. 10, 1345 (1996).

Science (1)

T. Verbiest, S. V. Elshocht, M. Kauranen, L. Hellemans, J. Snauwaert, C. Nuckolls, T. J. Katz, and A. Persoons, Science 282, 913 (1998).
[CrossRef] [PubMed]

Other (1)

S. Inoue, Video Microscopy (Plenum, New York, 1986).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

SHG images of two frames of Acetobacter in a 50µm stack, separated by 10 µm. Scale bar is 40 µm.

Fig. 2
Fig. 2

(a)–(c) SHG images from a 16µm-thick Valonia cell wall. The arrows point in the direction of the fibrils in the different lamellae. (d) z projection of 30 slices through the cell wall, showing all the lamellae. Scale bar is 40 µm.

Fig. 3
Fig. 3

SHG polarization dependence of fibrils in Valonia. (a),(b) Orthogonal excitations. The polarization angle was chosen to be optimal in (a). (c) Integrated SHG intensity as a function of laser polarization, where the data were taken every 4°, and the resulting fit to a cos2 θ distribution. Scale bar is 40 µm.

Fig. 4
Fig. 4

Enzymatic degradation kinetics of Valonia microfibrils by Aspergillis cellulase. The data and exponential fit are plotted and result in a time constant of 286 s R2=0.997.

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