Abstract
Classical fluorescence microscopy is a powerful technique to image biological specimen under close-to-native conditions, but light diffraction limits its optical resolution to 200–300 nm-two orders of magnitude worse than the size of biomolecules. Assuming single fluorescent emitters, the final image of the optical system can be described by a convolution with the point spread function (PSF) smearing out details below the size of the PSF. In mathematical terms, fluorescence microscopy produces bandlimited space-continuous images that can be recovered from their spatial samples under the conditions of the classical Shannon-Nyquist theorem. During the past two decades, several single molecule localization techniques have been established and these allow for the determination of molecular positions with sub-pixel accuracy. Without noise, single emitter positions can be recovered precisely – no matter how close they are. We review recent work on the computational resolution limit with a sharp phase transition between two scenarios: 1) where emitters are well-separated with respect to the bandlimit and can be recovered up to the noise level and 2) closely distributed emitters which results in a strong noise amplification in the worst case. We close by discussing additional pitfalls using single molecule localization techniques based on structured illumination.
Funding source: Volkswagen Foundation
Award Identifier / Grant number: Stability of Moment Problems and Super-Resolution
Funding source: Deutsche Forschungsgemeinschaft
Award Identifier / Grant number: CRC944
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Author contributions: All authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
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Research funding: This work was funded within the Collaborative Research Center 944 “Physiology and Dynamics of Cellular Microcompartments” (Project Z: Advanced imaging techniques) and by the Volkswagen Foundation project “Stability of Moment Problems and Super-Resolution Imaging”.
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Conflict of interest statement: The authors declare no conflicts of interest regarding this article.
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© 2023 Walter de Gruyter GmbH, Berlin/Boston
Artikel in diesem Heft
- Frontmatter
- Highlight: Physiology and Dynamics of Cellular Microcompartments
- Highlight: on the past and the future of cellular microcompartments
- Nuclear redox processes in land plant development and stress adaptation
- The readily retrievable pool of synaptic vesicles
- Loss of respiratory complex I subunit NDUFB10 affects complex I assembly and supercomplex formation
- Modulation of self-organizing circuits at deforming membranes by intracellular and extracellular factors
- Computational resolution in single molecule localization – impact of noise level and emitter density
- Setting up a data management infrastructure for bioimaging
- Molecular insights into endolysosomal microcompartment formation and maintenance
- The role of lysosomes in lipid homeostasis
- Membrane damage and repair: a thin line between life and death
- Neuronal stress granules as dynamic microcompartments: current concepts and open questions
- Molecular determinants of protein half-life in chloroplasts with focus on the Clp protease system
- Neprilysin 4: an essential peptidase with multifaceted physiological relevance
- Determinants of synergistic cell-cell interactions in bacteria
- Drosophila collagens in specialised extracellular matrices
Artikel in diesem Heft
- Frontmatter
- Highlight: Physiology and Dynamics of Cellular Microcompartments
- Highlight: on the past and the future of cellular microcompartments
- Nuclear redox processes in land plant development and stress adaptation
- The readily retrievable pool of synaptic vesicles
- Loss of respiratory complex I subunit NDUFB10 affects complex I assembly and supercomplex formation
- Modulation of self-organizing circuits at deforming membranes by intracellular and extracellular factors
- Computational resolution in single molecule localization – impact of noise level and emitter density
- Setting up a data management infrastructure for bioimaging
- Molecular insights into endolysosomal microcompartment formation and maintenance
- The role of lysosomes in lipid homeostasis
- Membrane damage and repair: a thin line between life and death
- Neuronal stress granules as dynamic microcompartments: current concepts and open questions
- Molecular determinants of protein half-life in chloroplasts with focus on the Clp protease system
- Neprilysin 4: an essential peptidase with multifaceted physiological relevance
- Determinants of synergistic cell-cell interactions in bacteria
- Drosophila collagens in specialised extracellular matrices