Abstract
THE use of fluorescence microscopy for investigating the three-dimensional structure of cells and tissue is of growing importance in cell biology, biophysics and biomedicine. Three-dimensional data are obtained by recording a series of images of the specimen as it is stepped through the focal plane of the microscope1–3. Whether by direct imaging or by confocal scanning4,5, diffraction effects and noise generally limit axial resolution to about 0.5 μm. Here we describe a fluorescence microscope in which axial resolution is increased to better than 0.05 μm by using the principle of standing-wave excitation of fluorescence. Standing waves formed by interference in laser illumination create an excitation field with closely spaced nodes and antinodes, allowing optical sectioning of the specimen at very high resolution. We use this technique to obtain images of actin fibres and filaments in fixed cells, actin single filaments in vitro and myosin II in a living cell.
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References
Fay, F. S., Fujiwara, K., Rees, D. D. & Fogarty, K. E. J. Cell Biol. 96, 783–795 (1983).
Agard, D. A. Rev. Biophys. Bioengn 13, 191–219 (1984).
Agard, D. A., Hiraoka, Y., Shaw, P. & Sedat, J. W. Meth. Cell Biol. 30, 353–377 (1989).
Wilson, T. & Sheppard, C. J. R. Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984).
Pawley, J. (ed) The Handbook of Biological Confocal Microscopy (IMR, Madison, 1989).
Carter, K. C., et al. Science 259, 1330–1335 (1993).
Sheppard, C. J. R. & Choudhury, A. Optica 24, 1051–1073 (1977).
Brakenhoff, G. J., Blom, P. & Barends, P. J Microsc. 117, 219–232 (1979).
Lanni, F. in Applications of Fluorescence in the Biomedical Sciences (eds Taylor, D. L. et al.) 505–521 (Liss, New York, 1986).
Lanni, F., Taylor, D. L. & Waggoner, A. S. US Patent No. 4,621,911 (1986).
Hell, S. & Stelzer, E. H. K. J. Opt. Soc. Am. A 9, 2159–2166 (1993).
Born, M. & Wolf, E. Principles of Optics 6th edn 439–441 (Pergamon, New York, 1980).
Giuliano, K. A. & Taylor, D. L. Cell Motil. Cytoskel. 16, 14–21 (1990).
Fisher, G. W., Conrad, P. A., DeBiasio, R. L. & Taylor, D. L. Cell Motil. Cytoskel. 11, 235–247 (1988)
Lanni, F., Waggoner, A. S. & Taylor, D. L. J. Cell Biol. 100, 1091–1102 (1985).
Bereiter-Hahn, J., Fox, C. H. & Thorell, B. J. Cell Biol. 82, 767–779 (1979).
Ross, K. F. A. & Gordon, R. E. J. Microsc. 128, 7–21 (1981).
DeBiasio, R. L., Wang, L.-L., Fisher, G. W. & Taylor, D. L. J. Cell Biol. 107, 2631–2645 (1988).
Small, J. V. J. Cell Biol. 91, 695–705 (1981).
Wang, Y. -L. J. Cell Biol. 101, 597–602 (1985).
Fan, J., Mansfield, S. G., Redmond, T., Gordon-Weeks, P. R. & Raper, J. A. J. Cell Biol. 121, 867–878 (1993).
Holmes, T. J. J. Opt. Soc. Am. A 5, 666–673 (1988).
Carrington, W. A. Soc. Photo-opt. Instrumentation Engng Proc. 1205, 72–83 (1990).
Podilchuk, C. I. & Mammone, R. J. J. Opt. Soc. Am. A 7, 517–521 (1990).
Koshy, M., Agard, D. A. & Sedat, J. W. Soc. Photo-opt. Instrumentation Engng Proc. 1205, 64–71 (1990).
Preza, C., Miller, M. I., Thomas, L. J. Jr., & McNally, J. G. J. Opt. Soc. Am. A 9, 219–228 (1992).
Kogelnik, H. & Li, T. Proc. Instn Electl Electron. Engrs 54, 1312–1329 (1966).
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Bailey, B., Farkas, D., Taylor, D. et al. Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature 366, 44–48 (1993). https://doi.org/10.1038/366044a0
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DOI: https://doi.org/10.1038/366044a0
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