Journal of Fluorescence

, Volume 21, Issue 3, pp 1075–1082

The Limitations of Nonlinear Fluorescence Effect in Super Resolution Saturated Structured Illumination Microscopy System

  • Aviram Gur
  • Zeev Zalevsky
  • Vicente Micó
  • Javier García
  • Dror Fixler
Original Paper

Abstract

Classically, optical systems are considered to have a fundamental resolution limit due to diffraction. Many strategies for improving both axial and lateral resolutions are based on a priori information about the input signal. These strategies lead to a numerical aperture improvement. However these are still limited by the wave nature of light. By using fluorescence technique one theoretically can reach unlimited resolution. The key point is to use the nonlinear dependence of the fluorescence emission rate on the intensity of the applied illumination. In this paper we present simulation as well as experimental results which show the advantage and the problems of using the nonlinear fluorescence effect in super resolution systems as well as discussing the nonlinear phenomena concerning the fluorescence process. The results show that the nonlinear fluorescence effect is accompanied by severe quenching, bleaching and saturation phenomena. As consequence, super resolution using saturated structured illumination method in living biological samples becomes severely restricted.

Keyword

Fluorescence Excitation power density Photo damage Super resolution Saturation Saturated structured illumination 

References

  1. 1.
    Abbe E (1873) Beitrage zur theorie des mikroskops und der mikroskopischen wahrnehmung. Arch Mikrosk Anat 9:413–468CrossRefGoogle Scholar
  2. 2.
    Toraldo Di Francia G (1955) Resolving power and information. J Opt Soc Am 45:497–501CrossRefGoogle Scholar
  3. 3.
    Toraldo Di Francia G (1969) Degrees of freedom of an image. J Opt Soc Am 59:799–804PubMedCrossRefGoogle Scholar
  4. 4.
    Lukosz W (1967) Optical systems with resolving powers exceeding the classical limits. J Opt Soc Am 56:1463–1472CrossRefGoogle Scholar
  5. 5.
    Lukosz W (1967) Optical systems with resolving powers exceeding the classical limits II. J Opt Soc Am 57:932–940CrossRefGoogle Scholar
  6. 6.
    Cox IJ, Sheppard JR (1986) Information capacity and resolution in an optical system. J Opt Soc Am A 3:1152–1158CrossRefGoogle Scholar
  7. 7.
    Zalevsky Z, Mendlovic D (2002) Optical super resolution. Springer.Google Scholar
  8. 8.
    Shemer A et al (1999) Superresolving optical system with time multiplexing and computer decoding. Appl Opt 38:7245–7251PubMedCrossRefGoogle Scholar
  9. 9.
    Semwogerere D, Weeks ER (2005) Confocal microscopy, encyclopedia of biomaterials and biomedical engineering. Taylor & FrancisGoogle Scholar
  10. 10.
    Hell SW, Kroug M (1995) Ground-state-depletion fluorescence microscopy: a concept for breaking the diffraction resolution limit. Appl Phys B 60:495–497CrossRefGoogle Scholar
  11. 11.
    Schönle A, Hänninen PE, Hell SW (1999) Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy. Ann Phys (Leipzig) 8:115–133CrossRefGoogle Scholar
  12. 12.
    Schönle A, Hell SW (1999) Far-field fluorescence microscopy with repetitive excitation. Eur Phys J D 6:283–290CrossRefGoogle Scholar
  13. 13.
    Heintzmann R, Cremer C (1999) Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proc SPIE 3568:185–195CrossRefGoogle Scholar
  14. 14.
    Frohn JT, Knapp HF, Stemmer A (2000) True optical resolution beyond the Rayleigh limit achieved by standing wave illumination. Proc Natl Acad Sci U S A 97:7232–7236PubMedCrossRefGoogle Scholar
  15. 15.
    Frohn JT, Knapp HF, Stemmer A (2001) Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation. Opt Lett 26:828–830PubMedCrossRefGoogle Scholar
  16. 16.
    Gustafsson MGL (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198:82–87PubMedCrossRefGoogle Scholar
  17. 17.
    Gustafsson MGL et al (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94:4957–4970PubMedCrossRefGoogle Scholar
  18. 18.
    Gustafsson MGL (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 102:13081–13086PubMedCrossRefGoogle Scholar
  19. 19.
    Betzig E et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645PubMedCrossRefGoogle Scholar
  20. 20.
    Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Meth 3:793–795CrossRefGoogle Scholar
  21. 21.
    Heintzmann R, Jovin TM, Cremer C (2002) Saturated patterned excitation microscopy - a concept for optical resolution improvement. J Opt Soc Am A 19:1599–1609CrossRefGoogle Scholar
  22. 22.
    Heintzmann R (2003) Saturated patterned excitation microscopy with two-dimensional excitation patterns. Micron 34:283–291PubMedCrossRefGoogle Scholar
  23. 23.
    Garcia J, Zalevsky Z, Fixler D (2005) Synthetic aperture super resolution by speckle pattern projection. Opt Exp 13:6073–6078CrossRefGoogle Scholar
  24. 24.
    Lindmo T, Steen HB (1977) Flow cytometric measurement of the polarization of fluorescence from intracellular fluorescein in mammalian cells. Biophys J 18:173–187PubMedCrossRefGoogle Scholar
  25. 25.
    Rotman B, Papermaster BW (1966) Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci 55:134–141PubMedCrossRefGoogle Scholar
  26. 26.
    Bloom JA, Webb WW (1984) Photodamage to intact erythrocyte membranes at high laser intensities: methods of assay and suppression. J Histochem Cytochem 32(6):608–616PubMedCrossRefGoogle Scholar
  27. 27.
    Lubart R et al (1993) Light effect on fibroblast proliferation. Laser Therapy 5:55–57Google Scholar
  28. 28.
    Sheetz MP, Koppel DE (1979) Membrane damage caused by irradiation of fluorescent concanavalin A. Proc Natl Acad Sci USA 76:3314–3317PubMedCrossRefGoogle Scholar
  29. 29.
    Shapiro HM (1983) Apparatus and method for killing unwanted cells, United States Patent 4395397Google Scholar
  30. 30.
    Deutsch M et al (2002) Fluorescence polarization as a functional parameter in monitoring living cells: theory and practice. J Fluoresc 12(1):25–44CrossRefGoogle Scholar
  31. 31.
    Keene JP, Hodgson BW (1980) A fluorescence polarization flow cytometer. Cytometry 1(2):118–126PubMedCrossRefGoogle Scholar
  32. 32.
    Pinkel D et al (1978) Fluorescence polarimeter for flow cytometry. Rev Sci Instrum 49(7):905–912PubMedCrossRefGoogle Scholar
  33. 33.
    Lang T, Rizzoli SO (2010) Membrane protein clusters at nanoscale resolution: more than pretty pictures. Physiology 25:116–124PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Aviram Gur
    • 1
  • Zeev Zalevsky
    • 1
  • Vicente Micó
    • 2
  • Javier García
    • 2
  • Dror Fixler
    • 1
  1. 1.School of engineeringBar Ilan UniversityRamat GanIsrael
  2. 2.Departamento de ÓpticaUniversitat de ValenciaBurjassotSpain

Personalised recommendations