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.
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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, 1029–1041
Odenheimer, J., Kreth, G., and Heermann, D. W. (2005) Dynamic simulation of active/inactive chromatin domains. J. Biol. Phys. 31, 351–363
Egner, A. and Hell, S. W. (2005) Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215
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, 1335–1337
Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., and Hell, S. W. (2006) Nanoscale resolution in GFP-based microscopy. Nature 440, 935–939
Westphal, V. and Hell, S. W. (2005) Nanoscale resolution in the focal plane of an optical microscope. Phys. Rev. Lett. 94, 143903
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, 147–156
Gustafsson, M. G. (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. USA 102, 13081–13086
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, 1599–1609
Frohn, J. T., Knapp, H. F., and Stemmer, A. (2001) Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation. Opt. Lett. 26, 828–830
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, 7232–7236
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, 118–136
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 York
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, 3511–3517
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, 1642–1645
Hess, S. T., Girirajan, T. P., and Mason, M. D. (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272
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, 44–48
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, 80–87
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, 7275–7283
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, 77–88
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, 8436–8443
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, 75–82
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, 4312–4318
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, 2449–2455
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, 2241–2253
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, 10–18
Miller, O. L., Jr. and Bakken, A. H. (1972) Morphological studies of transcription. Acta Endocrinol. Suppl. 168, 155–177
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, 1523–1536
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Birk, U.J., Baddeley, D., Cremer, C. (2008). Nanosizing by Spatially Modulated Illumination (SMI) Microscopy and Applications to the Nucleus. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 464. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-461-6_21
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DOI: https://doi.org/10.1007/978-1-60327-461-6_21
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