Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy
06 Jun 2008-Science (American Association for the Advancement of Science)-Vol. 320, Iss: 5881, pp 1332-1336
TL;DR: Three-dimensional structured illumination microscopy (3D-SIM) opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.
Abstract: Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.
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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 Multicolor Imaging o..."
...SSIM has only been demonstrated in two dimensions to date, while the nonsaturated SIM has provided ~100-nm lateral and ~300-nm axial resolution, allowing chromosomes and nuclear envelope structures to be better resolved compared to confocal microscopy (21)....
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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 or methods from "Subdiffraction Multicolor Imaging o..."
...One such example is the identification of interchromatin channels leading up to individual nuclear pores (Schermelleh et al., 2008)....
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...With three-dimensional SIM (3D-SIM), an additional twofold increase in the axial resolution can be achieved by generating an excitation light modulation along the z-axis using three-beam interference (Gustafsson et al., 2008; Schermelleh et al., 2008) and processing a z-stack of images accordingly....
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TL;DR: This Primer explains the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)PALM, and demonstrates how these approaches are beginning to provide new insights into cell biology, microbiology, and neurobiology.
1,056 citations
Cites background or methods from "Subdiffraction Multicolor Imaging o..."
...For example, studies with 3D SIM revealed morphological changes of the chromosomes and the nuclear lamin during early mitosis (Schermelleh et al., 2008)....
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...As an example, Figure 2D shows a three-color SIM image of DNA, lamin B, and nuclear pore complexes in the nucleus (Schermelleh et al., 2008)....
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...From Schermelleh et al., 2008....
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...Similar to the 2D counterpart, 3D SIM can double the resolution in all three dimensions, resulting in 100 nm resolution in the lateral directions and 300nm in the axial direction (Gustafsson et al., 2008; Schermelleh et al., 2008)....
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TL;DR: How the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells is outlined.
Abstract: Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed the DNA damage response (DDR). A common feature of DDR proteins is their mobilization in response to genotoxic stress. Here, we outline how the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells. Considerable advances have also been made in understanding the underlying molecular mechanisms for these events, with post-translational modifications of DDR factors being shown to play prominent roles in controlling the formation of foci in response to DNA-damaging agents. We review these regulatory mechanisms and discuss their biological significance to the DDR.
1,051 citations
Cites background from "Subdiffraction Multicolor Imaging o..."
...…further progress in the field is also likely to be triggered by further technical improvements, such as new superresolution microscopes and associated computational tools that will allow hitherto unattainable resolution of DDR foci in both three-dimensional space and time (Schermelleh et al. 2008)....
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...Thus, it seems that further progress in the field is also likely to be triggered by further technical improvements, such as new superresolution microscopes and associated computational tools that will allow hitherto unattainable resolution of DDR foci in both three-dimensional space and time (Schermelleh et al. 2008)....
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TL;DR: An overview of current super-resolution microscopy techniques is given and guidance on how best to use them to foster biological discovery is provided.
Abstract: Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
665 citations
References
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7,924 citations
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7,213 citations
01 Jan 1990
TL;DR: Methods for Three-Dimensional Imaging and Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen.
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4,121 citations
TL;DR: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide‐field fluorescence microscope with strikingly increased clarity compared to both conventional and confocal microscopes.
Abstract: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.
3,274 citations
TL;DR: Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.
Abstract: In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.
2,730 citations