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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TL;DR: In this article , a method for the preparation of microsphere/cellular lens array modules based on maskless lithography is proposed, where natural sedimentation principle is utilized to form microlens arrays by assembling SiO2 microspheres and MCF-7 cells on the hydrogel microporous module.
Abstract:
In the field of micro and nano optics, transparent dielectric microspheres and living biological cells have attracted more and more attention due to their optical imaging capabilities. However, it is difficult to integrate them directly onto optical systems because of their size and bioactivity limitations. Although multi-physics field methods based on optical tweezers and acoustic tweezers can manipulate microspheres and cells for imaging, they tend to have specific requirements for imaging samples. Here, we proposed a method for the preparation of microsphere/cellular lens array modules based on maskless lithography. Natural sedimentation principle is utilized to form microlens arrays by assembling SiO2 microspheres and MCF-7 cells on the hydrogel microporous module. The experimental and simulation results show that the SiO2 microsphere array embedded in the hydrogel still maintains its super-resolution imaging capability, and the MCF-7 cells can achieve the image magnification. As a result of the arrayed imaging, a larger viewing field can be obtained than that of individual microspheres or cells. The modular imaging method proposed in this paper is expected to be applied in the field of biophotonic devices and
TL;DR: Zhang et al. as mentioned in this paper developed zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary SR imaging conditions in an unsupervised manner.
Abstract: Computational super-resolution (SR) methods, including conventional analytical algorithms and deep learning models, have substantially improved optical microscopy. Among them, supervised deep neural networks have demonstrated outstanding SR performance, however, demanding abundant high-quality training data, which are laborious and even impractical to acquire due to the high dynamics of living cells. Here, we develop zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary SR imaging conditions in an unsupervised manner without the need for either ground truths or additional data acquisition. We demonstrate the versatile applicability of ZS-DeconvNet on multiple imaging modalities, including total internal reflection fluorescence microscopy, three-dimensional (3D) wide-field microscopy, confocal microscopy, lattice light-sheet microscopy, and multimodal structured illumination microscopy (SIM), which enables multi-color, long-term, super-resolution 2D/3D imaging of subcellular bioprocesses from mitotic single cells to multicellular embryos of mouse and C. elegans.
15 Mar 2023
TL;DR: In this paper , a time dependent likelihood distribution for analyzing time correlated single photon counting data from a four-pixel time-resolved single molecule localization microscopy experiment is discussed, by accounting for the probabilities to record photons from two emitters, background counts, and dark counts during two different time channels relative to each incident laser pulse in the experiment.
Abstract: A time-dependent likelihood distribution for analyzing time correlated single photon counting data from a four-pixel time-resolved single molecule localization microscopy experiment is discussed. It is generated by accounting for the probabilities to record photons from two emitters, background counts, and dark counts during two different time channels relative to each incident laser pulse in the experiment. Maximizing the distribution enables localization of each emitter in a dual emitting nanostructure based on the disparate photoluminescence lifetimes of the emitters, even when both emitters are simultaneously in an emissive state. The technique is demonstrated using simulated photon counting data from a hypothetical non-blinking dual-emitter nanostructure in which the distance between the two emitters is less than 10-nm.
TL;DR: These advances start with Grew's simple and—at the time—surprising realization that plant cells are as complex as animals cells, and that the different parts of the plant body indeed qualify to be called “organs”, then move on to the development of the groundbreaking “cell theory” in the mid‐19th century and the description of eu‐ and heterochromatin in the early 20th century.
Abstract: When the microscope was first introduced to scientists in the 17th century, it started a revolution. Suddenly, a whole new world, invisible to the naked eye, was opened to curious explorers. In response to this realization, Nehemiah Grew, an English plant anatomist and physiologist and one of the early microscopists, noted in 1682 “that Nothing hereof remains further to be known, is a Thought not well Calculated”. Since Grew made his observations, the microscope has undergone numerous variations, developing from early compound microscopes—hollow metal tubes with a lens on each end—to the modern, sophisticated, out‐of‐the‐box super‐resolution microscopes available to researchers today. In this Overview article, I describe these developments and discuss how each new and improved variant of the microscope led to major breakthroughs in the life sciences, with a focus on the plant field. These advances start with Grew's simple and—at the time—surprising realization that plant cells are as complex as animals cells, and that the different parts of the plant body indeed qualify to be called “organs”, then move on to the development of the groundbreaking “cell theory” in the mid‐19th century and the description of eu‐ and heterochromatin in the early 20th century, and finish with the precise localization of individual proteins in intact, living cells that we can perform today. Indeed, Grew was right; with ever‐increasing resolution, there really does not seem to be an end to what can be explored with a microscope. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.
References
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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。
18,940 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: A localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations), and results show good agreement with the derived precision equation.
2,390 citations
TL;DR: Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples, and a recently proposed method in which the nonlinearity arises from saturation of the excited state is experimentally demonstrated.
Abstract: Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arisesfromsaturationoftheexcitedstate.Thismethodcanbeused in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.
2,125 citations
TL;DR: The results strongly support a hand-over-hand model of motility, not an inchworm model, which moves processively on actin.
Abstract: Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of 1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 2x nm and 37 – 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model. Myosin V is a cargo-carrying processive motor
1,888 citations