Imaging intracellular fluorescent proteins at nanometer resolution.
15 Sep 2006-Science (American Association for the Advancement of Science)-Vol. 313, Iss: 5793, pp 1642-1645
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.
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28 Jul 2005
TL;DR: PfPMP1）与感染红细胞、树突状组胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作�ly.
Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。
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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.
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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.
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TL;DR: 3D stochastic optical reconstruction microscopy (STORM) is demonstrated by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy, allowing the 3D morphology of nanoscopic cellular structures to be resolved.
Abstract: Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.
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TL;DR: Optical antennas are devices that convert freely propagating optical radiation into localized energy, and vice versa as mentioned in this paper, and hold promise for enhancing the performance and efficiency of photodetection, light emission and sensing.
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References
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TL;DR: The near-field optical interaction between a sharp probe and a sample of interest can be exploited to image, spectroscopically probe, or modify surfaces at a resolution inaccessible by traditional far-field techniques, resulting in a technique of considerable versatility.
Abstract: The near-field optical interaction between a sharp probe and a sample of interest can be exploited to image, spectroscopically probe, or modify surfaces at a resolution (down to ∼12 nm) inaccessible by traditional far-field techniques. Many of the attractive features of conventional optics are retained, including noninvasiveness, reliability, and low cost. In addition, most optical contrast mechanisms can be extended to the near-field regime, resulting in a technique of considerable versatility. This versatility is demonstrated by several examples, such as the imaging of nanometric-scale features in mammalian tissue sections and the creation of ultrasmall, magneto-optic domains having implications for highdensity data storage. Although the technique may find uses in many diverse fields, two of the most exciting possibilities are localized optical spectroscopy of semiconductors and the fluorescence imaging of living cells.
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TL;DR: A photoactivatable variant of the Aequorea victoria green fluorescent protein is reported that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometers light and remains stable for days under aerobic conditions.
Abstract: We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.
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TL;DR: Individual carbocyanine dye molecules in a sub-monolayer spread have been imaged with near-field scanning optical microscopy and the orientation of each molecular dipole can be determined to map the electric field distribution in the near- field aperture with molecular spatial resolution.
Abstract: Individual carbocyanine dye molecules in a sub-monolayer spread have been imaged with near-field scanning optical microscopy. Molecules can be repeatedly detected and spatially localized (to ∼λ/50 where λ is the wavelength of light) with a sensitivity of at least 0.005 molecules/(Hz)1/2 and the orientation of each molecular dipole can be determined. This information is exploited to map the electric field distribution in the near-field aperture with molecular spatial resolution.
1,201 citations
TL;DR: It is shown that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated, suggesting that at least some vesicles constituents remain together during recycling.
Abstract: STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis
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TL;DR: The development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells are traced.
Abstract: The ability to visualize, track, and quantify molecules and events in living cells with high spatial and temporal resolution is essential for understanding biological systems. Only recently has it become feasible to carry out these tasks due to the advent of fluorescent protein technology. Here, we trace the development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells.
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