Three-dimensional Resolution Doubling in Wide-field Fluorescence Microscopy by Structured Illumination

Overview

Journal Biophys J

Publisher Cell Press

Specialty Biophysics

Date 2008 Mar 11

PMID 18326650

Citations 565

Authors

Mats G L Gustafsson

Lin Shao

Peter M Carlton

C J Rachel Wang

Inna N Golubovskaya

W Zacheus Cande

David A Agard

John W Sedat

Affiliations

Soon will be listed here.

Abstract

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.

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References

Dernburg A, Sedat J, Hawley R . Direct evidence of a role for heterochromatin in meiotic chromosome segregation. Cell. 1996; 86(1):135-46. DOI: 10.1016/s0092-8674(00)80084-7. View

Gustafsson M, Agard D, Sedat J . I5M: 3D widefield light microscopy with better than 100 nm axial resolution. J Microsc. 1999; 195(Pt 1):10-6. DOI: 10.1046/j.1365-2818.1999.00576.x. View

Carrington W, Lynch R, Moore E, Isenberg G, Fogarty K, Fay F . Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. Science. 1995; 268(5216):1483-7. DOI: 10.1126/science.7770772. View

Gustafsson M . Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 2000; 198(Pt 2):82-7. DOI: 10.1046/j.1365-2818.2000.00710.x. View

Chung E, Kim D, Cui Y, Kim Y, So P . Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens. Biophys J. 2007; 93(5):1747-57. PMC: 1948056. DOI: 10.1529/biophysj.106.097907. View

Agard D, Hiraoka Y, Shaw P, Sedat J . Fluorescence microscopy in three dimensions. Methods Cell Biol. 1989; 30:353-77. DOI: 10.1016/s0091-679x(08)60986-3. View

Armstrong S, Caryl A, Jones G, Franklin F . Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica. J Cell Sci. 2002; 115(Pt 18):3645-55. DOI: 10.1242/jcs.00048. View

Gustafsson M . Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A. 2005; 102(37):13081-6. PMC: 1201569. DOI: 10.1073/pnas.0406877102. View

Schwentker M, Bock H, Hofmann M, Jakobs S, Bewersdorf J, Eggeling C . Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching. Microsc Res Tech. 2007; 70(3):269-80. DOI: 10.1002/jemt.20443. View

10.

Golubovskaya I, Hamant O, Timofejeva L, Wang C, Braun D, Meeley R . Alleles of afd1 dissect REC8 functions during meiotic prophase I. J Cell Sci. 2006; 119(Pt 16):3306-15. DOI: 10.1242/jcs.03054. View

11.

Hess S, Girirajan T, Mason M . Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J. 2006; 91(11):4258-72. PMC: 1635685. DOI: 10.1529/biophysj.106.091116. View

12.

Frohn J, Knapp H, Stemmer A . Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation. Opt Lett. 2007; 26(11):828-30. DOI: 10.1364/ol.26.000828. View

13.

Heintzmann R, Jovin T, Cremer C . Saturated patterned excitation microscopy--a concept for optical resolution improvement. J Opt Soc Am A Opt Image Sci Vis. 2002; 19(8):1599-609. DOI: 10.1364/josaa.19.001599. View

14.

Rust M, Bates M, Zhuang X . Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006; 3(10):793-5. PMC: 2700296. DOI: 10.1038/nmeth929. View

15.

Weiner O, Rentel M, Ott A, Brown G, Jedrychowski M, Yaffe M . Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis. PLoS Biol. 2006; 4(2):e38. PMC: 1334198. DOI: 10.1371/journal.pbio.0040038. View

16.

Bailey B, Farkas D, Taylor D, Lanni F . Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature. 1993; 366(6450):44-8. DOI: 10.1038/366044a0. View

17.

Klar T, Jakobs S, Dyba M, Egner A, Hell S . Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci U S A. 2000; 97(15):8206-10. PMC: 26924. DOI: 10.1073/pnas.97.15.8206. View

18.

Page S, Hawley R . The genetics and molecular biology of the synaptonemal complex. Annu Rev Cell Dev Biol. 2004; 20:525-58. DOI: 10.1146/annurev.cellbio.19.111301.155141. View

19.

Frohn J, Knapp H, Stemmer A . True optical resolution beyond the Rayleigh limit achieved by standing wave illumination. Proc Natl Acad Sci U S A. 2000; 97(13):7232-6. PMC: 16528. DOI: 10.1073/pnas.130181797. View

20.

Golubovskaya I, Harper L, Pawlowski W, Schichnes D, Cande W . The pam1 gene is required for meiotic bouquet formation and efficient homologous synapsis in maize (Zea mays L.). Genetics. 2003; 162(4):1979-93. PMC: 1462385. DOI: 10.1093/genetics/162.4.1979. View