Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission
Open accessAbstract
The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90–110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.
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Scanning the lasers with very small steps over the sample improves the resolution of the final image up to 50 nm.
“Therefore, scanning the lasers with very small steps over the sample improves the resolution of the final image up to 50 nm (Klar et al., 2000; Hell, 2007).”
From Barriers in the brain: resolving dendritic spine morphology and compartmentalization (2014) · cites this paper as Klar et al., 2000
The passage discussing Fig. 2 (page not supplied) says 40–50 nm resolution could be obtained by deconvolution, not by scanning in very small steps. The paper reports achieved resolution around 90–110 nm and attributes the improvement to stimulated emission depletion. Its mention of a stage with 5-nm precision concerns measuring an excitation spot, not improving the final image to 50 nm. (LLM, conf=0.88)
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Research works printed but not linked: 18
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- Authors
- Klar, T. A., Jakobs, S., Dyba, M., Egner, A., & Hell, S. W.
- Affiliations
- Max Planck Institute for Biophysical Chemistry (DE)
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2000-07-18
- Volume
- 97
- Issue
- 15
- Pages
- 8206-8210
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC26924
- OpenAlex
- W1978249718
- Cited by
- 1,869
- References
- 0
- Citations by year
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- FWCI
- 17.93
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- GRO (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Physical Sciences › Engineering › Biomedical Engineering › Near-Field Optical Microscopy
- Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Laser-Matter Interactions and Applications
- Keywords
- stimulated emission, fluorescence microscopy, diffraction limit, focal spot size, stimulated emission depletion, fluorescence quenching, sub-diffraction resolution, single-molecule spectroscopy, live-cell imaging
- MeSH terms
- Quaternary Ammonium Compounds, Escherichia coli, Fluorescent Dyes, Microscopy, Fluorescence, Organic Chemicals, Pyridinium Compounds, Saccharomyces cerevisiae, Nonlinear Dynamics
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