Microscopy and Its Focal Switch

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2009 Jan 1

PMID 19116611

Citations 253

Authors

Stefan W Hell

Affiliations

Soon will be listed here.

Abstract

Until not very long ago, it was widely accepted that lens-based (far-field) optical microscopes cannot visualize details much finer than about half the wavelength of light. The advent of viable physical concepts for overcoming the limiting role of diffraction in the early 1990s set off a quest that has led to readily applicable and widely accessible fluorescence microscopes with nanoscale spatial resolution. Here I discuss the principles of these methods together with their differences in implementation and operation. Finally, I outline potential developments.

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References

Folling J, Bossi M, Bock H, Medda R, Wurm C, Hein B . Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods. 2008; 5(11):943-5. DOI: 10.1038/nmeth.1257. View

Schonle A, Hell S . Fluorescence nanoscopy goes multicolor. Nat Biotechnol. 2007; 25(11):1234-5. DOI: 10.1038/nbt1107-1234. View

Cremer C, Cremer T . Considerations on a laser-scanning-microscope with high resolution and depth of field. Microsc Acta. 1978; 81(1):31-44. View

Hell S, Wichmann J . Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett. 2009; 19(11):780-2. DOI: 10.1364/ol.19.000780. View

Huang B, Wang W, Bates M, Zhuang X . Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science. 2008; 319(5864):810-3. PMC: 2633023. DOI: 10.1126/science.1153529. View

Shroff H, Galbraith C, Galbraith J, Betzig E . Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods. 2008; 5(5):417-23. PMC: 5225950. DOI: 10.1038/nmeth.1202. 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

Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B, Mukherjee A . Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl. 2008; 47(33):6172-6. DOI: 10.1002/anie.200802376. View

Hell S . Toward fluorescence nanoscopy. Nat Biotechnol. 2003; 21(11):1347-55. DOI: 10.1038/nbt895. View

10.

Egner A, Geisler C, Von Middendorff C, Bock H, Wenzel D, Medda R . Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys J. 2007; 93(9):3285-90. PMC: 2025649. DOI: 10.1529/biophysj.107.112201. View

11.

Dedecker P, Hotta J, Flors C, Sliwa M, Uji-I H, Roeffaers M . Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa. J Am Chem Soc. 2007; 129(51):16132-41. DOI: 10.1021/ja076128z. View

12.

Hofmann M, Eggeling C, Jakobs S, Hell S . Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci U S A. 2005; 102(49):17565-9. PMC: 1308899. DOI: 10.1073/pnas.0506010102. View

13.

Heintzmann R, Ficz G . Breaking the resolution limit in light microscopy. Brief Funct Genomic Proteomic. 2006; 5(4):289-301. DOI: 10.1093/bfgp/ell036. View

14.

Weiss S . Fluorescence spectroscopy of single biomolecules. Science. 1999; 283(5408):1676-83. DOI: 10.1126/science.283.5408.1676. View

15.

Von Middendorff C, Egner A, Geisler C, Hell S, Schonle A . Isotropic 3D Nanoscopy based on single emitter switching. Opt Express. 2008; 16(25):20774-88. DOI: 10.1364/oe.16.020774. View

16.

Egner A, Hell S . Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 2005; 15(4):207-15. DOI: 10.1016/j.tcb.2005.02.003. View

17.

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

18.

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

19.

Bretschneider S, Eggeling C, Hell S . Breaking the diffraction barrier in fluorescence microscopy by optical shelving. Phys Rev Lett. 2007; 98(21):218103. DOI: 10.1103/PhysRevLett.98.218103. View

20.

Moerner , Kador . Optical detection and spectroscopy of single molecules in a solid. Phys Rev Lett. 1989; 62(21):2535-2538. DOI: 10.1103/PhysRevLett.62.2535. View