Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes†
About: This article is published in Angewandte Chemie.The article was published on 2008-08-04. It has received 1692 citations till now. The article focuses on the topics: Photoactivated localization microscopy & Fluorescence-lifetime imaging microscopy.
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TL;DR: It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.
Abstract: Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.
1,534 citations
Cites background from "Subdiffraction-Resolution Fluoresce..."
...5, and Cy7 dyes can also be spontaneously activated and deactivated by the same imaging laser (23) (for example 647 nm) without the need of activators but emit light of different wavelenths ranging from red to near IR, providing the possibility of multicolor imaging by distinguishing emission color only....
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TL;DR: These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules and have created unprecedented new possibilities to investigate the structure and function of cells.
Abstract: For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.
1,204 citations
Cites background from "Subdiffraction-Resolution Fluoresce..."
...Variations of this method have been published by numerous groups adding new acronyms, such as SPDM (spectral precision distance measurement; Bornfleth et al., 1998; Lemmer et al., 2009), PALMIRA (PALM with independently running acquisition; Egner et al., 2007), GSDIM (ground state depletion and individual molecule return; Fölling et al., 2008b), or dSTORM (direct STORM; Heilemann et al., 2008)....
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...…(spectral precision distance measurement; Bornfleth et al., 1998; Lemmer et al., 2009), PALMIRA (PALM with independently running acquisition; Egner et al., 2007), GSDIM (ground state depletion and individual molecule return; Fölling et al., 2008b), or dSTORM (direct STORM; Heilemann et al., 2008)....
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TL;DR: This analysis quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images, providing guidelines for characterization ofsuper-resolution probes and a resource for selecting probes based on performance.
Abstract: One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resolution imaging.
1,188 citations
TL;DR: Inspired by molecular modeling, the N,N-dimethylamino substituents in tetramethylrhodamine are replaced with four-membered azetidine rings, which doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging.
Abstract: Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
1,140 citations
TL;DR: The principles of these methods together with their differences in implementation and operation are discussed, and potential developments are outlined.
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.
978 citations
References
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TL;DR: This work introduced a method for optically imaging intracellular proteins at nanometer spatial resolution and used this method to image specific target proteins in thin sections of lysosomes and mitochondria and in fixed whole cells to image retroviral protein Gag at the plasma membrane.
Abstract: We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
7,924 citations
TL;DR: A high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores that can, in principle, reach molecular-scale resolution is developed.
Abstract: We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.
7,213 citations
TL;DR: A new type of scanning fluorescence microscope capable of resolving 35 nm in the far field is proposed, overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function.
Abstract: We propose a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field. We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function. In contrast to near-field scanning optical microscopy, this method can produce three-dimensional images of translucent specimens.
5,110 citations
Journal Article•
TL;DR: In this paper, the authors proposed a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point spread function.
Abstract: We propose a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field. We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function. In contrast to near-field scanning optical microscopy, this method can produce three-dimensional images of translucent specimens.
3,987 citations
TL;DR: A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit, and suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.
3,437 citations