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The Nucleus pp 389-401 | Cite as

Nanosizing by Spatially Modulated Illumination (SMI) Microscopy and Applications to the Nucleus

  • Udo J. Birk
  • David Baddeley
  • Christoph Cremer
Part of the Methods in Molecular Biology book series (MIMB, volume 464)

Abstract

In this chapter we present the method of spatially modulated illumination (SMI) microscopy, a (far-field) fluorescence microscopy technique featuring structured illumination obtained via a standing wave field laser excitation pattern. While this method does not provide higher optical resolution, it has been proven a highly valuable tool to access structural parameters of fluorescently labeled macromolecular structures in cells. SMI microscopy has been used to measure relative positions with a reproducibility of <2 nm between fluorescing objects. Among others, we have measured size distributions of protein clusters with an accuracy much better than the resolution achievable e.g. in confocal microscopy. The advantages of the SMI microscope over other (ultra-)high resolution light microscopes are its easy sample preparation and microscope handling as well as the comparably fast acquisition times and large fields of view.

Keywords

SMI nanosizing Structured illumination Sizes and distances Transcription factories Fluorescence microscopy 

References

  1. 1.
    O’Brien, T. P., Bult, C. J., Cremer, C., Grunze, M., Knowles, B. B., Langowski, J., McNally, J., Pederson, T., Politz, J. C., Pombo, A., Schmahl, G., Spatz, J. P., and van Driel, R. (2003) Genome function and nuclear architecture: from gene expression to nanoscience. Genome Res. 13, 1029–1041PubMedCrossRefGoogle Scholar
  2. 2.
    Odenheimer, J., Kreth, G., and Heermann, D. W. (2005) Dynamic simulation of active/inactive chromatin domains. J. Biol. Phys. 31, 351–363PubMedCrossRefGoogle Scholar
  3. 3.
    Egner, A. and Hell, S. W. (2005) Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215PubMedCrossRefGoogle Scholar
  4. 4.
    Hell, S. W., Lindek, S., Cremer, C., and Stelzer, E. H. K. (1994) Measurement of the 4Pi-confocal point spread function proves 75 nm resolution. Appl. Phys. Lett. 64, 1335–1337CrossRefGoogle Scholar
  5. 5.
    Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., and Hell, S. W. (2006) Nanoscale resolution in GFP-based microscopy. Nature 440, 935–939PubMedCrossRefGoogle Scholar
  6. 6.
    Westphal, V. and Hell, S. W. (2005) Nanoscale resolution in the focal plane of an optical microscope. Phys. Rev. Lett. 94, 143903PubMedCrossRefGoogle Scholar
  7. 7.
    Gustafsson, M. G. L., Agard, D. A., and Sedat, J. W. (1995) Seven-fold improvement of axial resolution in 3-D widefield microscopy using two objective lenses. Proc. SPIE 2412, 147–156CrossRefGoogle Scholar
  8. 8.
    Gustafsson, M. G. (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. USA 102, 13081–13086PubMedCrossRefGoogle Scholar
  9. 9.
    Heintzmann, R., Jovin, T. M., and Cremer, C. (2002) Saturated patterned excitation microscopy (SPEM)–a novel concept for optical resolution improvement. J. Opt. Soc. Am. A. Opt. Image Sci. Vis. 19, 1599–1609PubMedCrossRefGoogle Scholar
  10. 10.
    Frohn, J. T., Knapp, H. F., and Stemmer, A. (2001) Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation. Opt. Lett. 26, 828–830PubMedCrossRefGoogle Scholar
  11. 11.
    Frohn, J. T., Knapp, H. F., and Stemmer, A. (2000) True optical resolution beyond the Rayleigh limit achieved by standing wave illumination. Proc. Natl. Acad. Sci. USA 97, 7232–7236PubMedCrossRefGoogle Scholar
  12. 12.
    Bornfleth, H., Sätzler, K., Eils, R., and Cremer, C. (1998) High precision distance measurements and volume-conserving segmentation of objects near and below the resolution limit in three-dimensional confocal fluorescence microscopy. J. Microsc. 189, 118–136CrossRefGoogle Scholar
  13. 13.
    Cremer, C., Edelmann, P., Bornfleth, H., Luz, H., Kreth, G., Münch, H., and Hausmann, M. (1999) in Handbook of computer vision and applications, Vol. 3 (Jahne, B., Hau³ecker, H. and Gei³ler, P., Eds.), pp. 839–857, Academic Press, San Diego, New YorkGoogle Scholar
  14. 14.
    Heilemann, M., Herten, D. P., Heintzmann, R., Cremer, C., Muller, C., Tinnefeld, P., Weston, K. D., Wolfrum, J., and Sauer, M. (2002) High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy. Anal. Chem. 74, 3511–3517PubMedCrossRefGoogle Scholar
  15. 15.
    Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J., and Hess, H. F. (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645PubMedCrossRefGoogle Scholar
  16. 16.
    Hess, S. T., Girirajan, T. P., and Mason, M. D. (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272PubMedCrossRefGoogle Scholar
  17. 17.
    Bailey, B., Farkas, D. L., Taylor, D. L., and Lanni, F. (1993) Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature 366, 44–48PubMedCrossRefGoogle Scholar
  18. 18.
    Albrecht, B., Schweitzer, A., Failla, A. V., Edelmann, P., and Cremer, C. (2002) Spatially modulated illumination (SMI) microscopy allows axial distance resolution in the nanometer range. Appl. Opt. 41, 80–87PubMedCrossRefGoogle Scholar
  19. 19.
    Failla, A. V., Spoeri, U., Albrecht, B., Kroll, A., and Cremer, C. (2002) Nanosizing of fluorescent objects by spatially modulated illumination microscopy. Appl. Opt. 41, 7275–7283PubMedCrossRefGoogle Scholar
  20. 20.
    Failla, A. V., Albrecht, B., Spöri, U., Schweitzer, A., Kroll, A., Hildenbrand, G., Bach, M., and Cremer, C. (2003) Nanostructure analysis using spatially modulated illumination microscopy. Com-PlexUs 1, 77–88Google Scholar
  21. 21.
    Spöri, U., Failla, A. V., and Cremer, C. (2004) Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy. J. Appl. Phys. 95, 8436–8443CrossRefGoogle Scholar
  22. 22.
    Mathée, H., Baddeley, D., Wotzlaw, C., Fandrey, J., Cremer, C., and Birk, U. (2006) Nanostructure of specific chromatin regions and nuclear complexes. Histochem. Cell Biol. 125, 75–82PubMedCrossRefGoogle Scholar
  23. 23.
    Hildenbrand, G., Rapp, A., Spoeri, U., Wagner, C., Cremer, C., and Hausmann, M. Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy. (2005) Biophys. J. 88, 4312–4318PubMedCrossRefGoogle Scholar
  24. 24.
    Martin, S., Failla, A. V., Spori, U., Cremer, C., and Pombo, A. (2004) Measuring the size of biological nanostructures with spatially modulated illumination microscopy. Mol. Biol. Cell 15, 2449–2455PubMedCrossRefGoogle Scholar
  25. 25.
    Pombo, A., Jackson, D. A., Hollinshead, M., Wang, Z., Roeder, R. G., and Cook, P. R. (1999) Regional specialization in human nuclei: visualization of discrete sites of transcription by RNA polymerase III. EMBO J. 18, 2241–2253PubMedCrossRefGoogle Scholar
  26. 26.
    Wansink, D. G., Sibon, O. C., Cremers, F. F., van Driel, R., and de Jong, L. (1996) Ultrastructural localization of active genes in nuclei of A431 cells. J. Cell. Biochem. 62, 10–18PubMedCrossRefGoogle Scholar
  27. 27.
    Miller, O. L., Jr. and Bakken, A. H. (1972) Morphological studies of transcription. Acta Endocrinol. Suppl. 168, 155–177Google Scholar
  28. 28.
    Jackson, D. A., Iborra, F. J., Manders, E. M., and Cook, P. R. (1998) Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei. Mol. Biol. Cell 9, 1523–1536PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Udo J. Birk
    • 1
  • David Baddeley
    • 1
  • Christoph Cremer
    • 1
  1. 1.Kirchhoff Institut für PhysikUniversität HeidelbergHeidelbergGermany

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