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Subdiffraction resolution in far-field fluorescence microscopy
Abstract
We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited molecules from the outer part of the focus through stimulated emission. This results in a subdiffraction-sized effective point-spread function. For a 1.4 aperture and a 388-nm excitation wavelength spatial resolution is increased from 150 +/- 8 nm to 106 +/- 8 nm with a single offset beam. Superior lateral resolution is demonstrated by separation of adjacent Pyridine 2 nanocrystals that are otherwise indiscernible.
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Citations
Cited by
Welsh, J. A., Goberdhan, D. C. I., O'Driscoll, L., Buzas, E. I., Blenkiron, C., Bussolati, B., Cai, H., Di Vizio, D., Driedonks, T. A. P., Erdbrügger, U., et al. (2024) · Journal of Extracellular Vesicles · doi:10.1002/jev2.12404 · Cited by 4,663
Gustafsson, M. G. L. (2000) · Journal of Microscopy · doi:10.1046/j.1365-2818.2000.00710.x · Cited by 3,786
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission
Klar, T. A., Jakobs, S., Dyba, M., Egner, A., & Hell, S. W. (2000) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.97.15.8206 · Cited by 1,869
Self-citationSuper-resolution fluorescence microscopy
Huang, B., Bates, M., & Zhuang, X. (2009) · Annual Review of Biochemistry · doi:10.1146/annurev.biochem.77.061906.092014 · Cited by 1,657
Macromolecular-scale resolution in biological fluorescence microscopy
Donnert, G., Keller, J., Medda, R., Andrei, M. A., Rizzoli, S. O., Lührmann, R., Jahn, R., Eggeling, C., & Hell, S. W. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0604965103 · Cited by 527
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Metadata
- Authors
- Klar, T. A., & Hell, S. W.
- Venue
- Optics Letters
- Publisher
- Optica Publishing Group
- Published
- 1999-07-15
- Volume
- 24
- Issue
- 14
- Pages
- 954
- ISSN
- 0146-9592 (ISSN-L)
- 1539-4794
- OpenAlex
- W1965238080
- Cited by
- 864
- References
- 0
- FWCI
- 5.90
- Type
- journal article
- Language
- en
- Open access
- closed
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- Klar, T. A., & Hell, S. W. (1999). Subdiffraction resolution in far-field fluorescence microscopy. Optics Letters. https://doi.org/10.1364/ol.24.000954
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.
