OSA's Digital Library

Applied Optics

Applied Optics


  • Vol. 41, Iss. 34 — Dec. 2, 2002
  • pp: 7275–7283

Nanosizing of fluorescent objects by spatially modulated illumination microscopy

Antonio Virgilio Failla, Udo Spoeri, Benno Albrecht, Alexander Kroll, and Christoph Cremer  »View Author Affiliations

Applied Optics, Vol. 41, Issue 34, pp. 7275-7283 (2002)

View Full Text Article

Enhanced HTML    Acrobat PDF (184 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



A new approach to measuring the sizes of small fluorescent objects by use of spatially modulated illumination (SMI) far-field light microscopy is presented. This method is based on SMI measurements combined with a new SMI virtual microscopy (VIM) data analysis calibration algorithm. Here, experimental SMI measurements of fluorescent objects with known diameter (size) were made. From the SMI data obtained, the size was determined in an independent way by use of the SMI VIM algorithm. The results showed that with SMI microscopy in combination with SMI VIM calibration, subwavelength object size measurements as small as 40 nm are experimentally feasible with high accuracy.

© 2002 Optical Society of America

OCIS Codes
(100.0100) Image processing : Image processing
(110.0180) Imaging systems : Microscopy
(180.0180) Microscopy : Microscopy
(180.2520) Microscopy : Fluorescence microscopy
(180.3170) Microscopy : Interference microscopy
(180.6900) Microscopy : Three-dimensional microscopy

Original Manuscript: February 20, 2002
Revised Manuscript: August 13, 2002
Published: December 1, 2002

Antonio Virgilio Failla, Udo Spoeri, Benno Albrecht, Alexander Kroll, and Christoph Cremer, "Nanosizing of fluorescent objects by spatially modulated illumination microscopy," Appl. Opt. 41, 7275-7283 (2002)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. G. Blobel, “Gene gating: a hypothesis,” Proc. Natl. Acad. Sci. USA 82, 8527–8530 (1985). [CrossRef] [PubMed]
  2. T. Cremer, A. Kurz, R. Zirbel, S. Dietzel, B. Rinke, E. Schröck, M. R. Speicher, U. Mathieu, A. Jauch, P. Emmerich, H. Scherthan, T. Ried, C. Cremer, P. Lichter, “Role of chromosome territories in the functional compartmentalization of the cell nucleus,” Cold Spring Harbor Symp. Quant. Biol. 58, 777–792 (1993). [CrossRef] [PubMed]
  3. A. I. Lamond, W. C. Earnshaw, “Structure and function in the nucleus,” Science. 280, 547–553 (1996). [CrossRef]
  4. T. Cremer, C. Cremer, “Nuclear architecture and gene regulation in mammalian cells,” Nature Rev. Genetics 4, 292–301 (2001). [CrossRef]
  5. D. L. Spector, “Nuclear organization and gene expression,” Exp. Cell. Res. 229, 189–197 (1996). [CrossRef] [PubMed]
  6. P. R. Cook, “The organization of replication and transcription,” Science 284, 1790–1795 (1999). [CrossRef] [PubMed]
  7. A. Pombo, D. A. Jackson, F. J. Iborra, M. Hollinshead, H. Kimura, K. Sugaya, P. R. Cook, “Transcription factories,” in Proceedings of Twelfth European Congress on Electron Microscope, Volume IBiological Sciences (n.p., 2000), pp. B461–B464.
  8. A. Pombo, P. Cuello, W. Schul, J.-B. Yoon, R. G. Roeder, P. R. Cook, S. Murphy, “Regional and temporal specialization in the nucleus: a transcriptionally-active nuclear domain rich in PTF, Oct1 and Pika antigens associates with specific chromosomes early in the cell cycle,” EMBO J. 17, 1768–1778 (1998). [CrossRef] [PubMed]
  9. A. Pombo, M. Hollinshead, P. R. Cook, “Bridging the resolution gap: imaging the same transcription factories in cryosections by light and electron microscopy,” Histochem. Cytochem. 47, 471–480 (1999). [CrossRef]
  10. A. Esa, P. Edelmann, A. L. Trakthenbrot, N. Amariglio, G. Rechavi, M. Hausmann, C. Cremer, “3D-spectral precision distance microscopy (SPDM) of chromatin nanostructures after triple-color labeling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome,” J. Microsc. 199, 96–105 (2000). [CrossRef] [PubMed]
  11. T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemia, A. P. Alivistatos, S. Weiss, “Ultrahigh-resolution multicolor colocalization of single fluorescent probes,” Proc. Natl. Acad. Sci. USA. 97, 9461–9466 (2000). [CrossRef] [PubMed]
  12. H. Bornfleth, K. Sätzler, R. Eils, C. Cremer, “High precision distance measurements and volume-conserving segmentation of objects near and below the resolution in three-dimensional confocal fluorescence microscopy,” J. Microsc. 189, 118–136 (1998). [CrossRef]
  13. B. Bailey, D. L. Farkas, D. L. Taylor, F. Lanni, “Enhancement of axial resolution in fluorescence microscopy by standing wave excitation,” Nature 366, 44–48 (1993). [CrossRef] [PubMed]
  14. B. Schneider, J. Bradl, I. Kirsten, M. Hausmann, C. Cremer, “High precision localization of fluorescent targets in the nanometer range by spatially modulated excitation fluorescence microscopy,” in Fluorescence Microscopy & Fluorescence Probes, J. Slavik, ed. (Plenum, New York, 1998), Vol. 2, pp. 63–68.
  15. A. V. Failla, C. Cremer, “Virtual spatially modulated illumination microscopy predicts nanometer precision of axial distance measurements,” in Optical Diagnostics of Living Cells IV, D. L. Farkas, R. C. Leif, eds., Proc. SPIE4260, 120–126 (2001).
  16. B. Albrecht, A. V. Failla, A. Schweitzer, C. Cremer, “Spatially modulated illumination microscopy allows axial distance resolution near one nanometer,” Appl. Opt. 41, 80–87 (2002). [CrossRef] [PubMed]
  17. B. Albrecht, R. Heintzmann, C. Cremer, “Online visualization of axial intensity distribution in spatially modulated illumination microscopy,” in Optical Diagnostics of Living Cells IV, D. L. Farkas, R. C. Leif, eds., Proc. SPIE4260, 127–140 (2001).
  18. S. W. Hell, S. Lindek, E. H. K. Stelzer, “Enhancing the axial resolution in far-field light microscopy two photon 4Pi confocal fluorescence microscopy,” J. Mod. Opt. 41, 675–681 (1994). [CrossRef]
  19. S. W. Hell, E. H. K. Stelzer, S. Lindek, C. Cremer, “Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy,” Opt. Lett. 19, 222–224 (1994). [CrossRef] [PubMed]
  20. S. W. Hell, S. Lindek, C. Cremer, E. H. K. Stelzer, “Measurement of the 4pi-confocal point spread function proves 75 nm axial resolution,” Appl. Phys. Lett. 64, 1335–1337 (1994). [CrossRef]
  21. S. Lindek, E. H. K. Stelzer, C. Cremer, “Confocal theta fluorescence microscopy with annular aperture,” Appl. Opt. 35, 126–130 (1996). [CrossRef] [PubMed]
  22. T. A. Klar, S. Jacobs, M. Dyba, A. Egner, S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. USA. 87, 8206–8210 (2000). [CrossRef]
  23. M. G. Gustafsson, D. A. Agard, J. W. Sedat, “Sevenfold improvement of axial resolution in 3D wide field microscopy using two objective lenses,” in Three-Dimensional Microscopy: Image Acquisition and Processing II, T. Wilson, C. J. Cogswell, eds, Proc. SPIE2412, 147–156 (1995).
  24. A. M. van Oijen, J. Köhler, J. Schmidt, M. Müller, G. J. Brakenhoff, “3-Dimensional super-resolution by spectrally selective imaging,” Chem. Phys. Lett. 292, 183–187 (1998). [CrossRef]
  25. J. T. Frohn, H. F. Knapp, A. Stemmer, “True optical resolution beyond the Rayleigh limit achieved by standing wave illumination,” Proc. Natl. Acad. Sci. USA 97, 7232–7236 (2000). [CrossRef] [PubMed]
  26. J. T. Frohn, H. F. Knapp, A. Stemmer, “Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation,” Opt. Lett. 26, 828–830 (2001). [CrossRef]
  27. A. V. Failla, A. Cavallo, C. Cremer, “Subwavelength size determination using SMI virtual microscopy,” Appl. Opt. 41, 6651–6659 (2002). [CrossRef] [PubMed]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited