- 标题
- 摘要
- 关键词
- 实验方案
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Photon Counting - Fundamentals and Applications || Detectors for Super-Resolution & Single-Molecule Fluorescence Microscopies
摘要： The resolution of light microscopy was thought to be limited to 250–300 nanometers based on the work of Ernest Abbe. This Abbe diffraction limit was believed to be insurmountable until the invention of Super-resolution microscopic techniques in the late 20th century. These techniques remove this limit and have provided unprecedented detail of cellular structures and dynamics down to several nanometers. An emerging goal in this field is to quantitatively measure individual molecules. Measurement of single-molecule dynamics, such as diffusion coefficients and complex stoichiometries, can be accomplished using fluorescence fluctuation techniques to reveal nanosecond-to-microsecond temporal reactions. These powerful complimentary experimental approaches are made possible by sensitive low-light photodetectors. In this chapter, an overview of the principles of super-resolution and single-molecule microscopies are provided. The different types of photodetectors employed in these techniques are explained. In addition, the advantages and disadvantages for these detectors are discussed, as well as the development of next generation detectors. Finally, example super-resolution and single-molecule cellular studies that take advantage of these detector technologies are presented.
关键词： biophysical techniques,STORM,nanoscopy,STED,protein dynamics,palm,spectroscopy,molecular brightness,fluorescence fluctuation
更新于2025-09-23 15:23:52
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Brain Storm Optimization Graph Theory (BSOGT) and Energy Resource Aware Virtual Network Mapping (ERVNM) for Medical Image System in Cloud
摘要： With the development of Internet and the make use of Internet for medical information, the demand for huge scale and reliable managing medical information has brought out the huge scale Internet data centers. This work that has been presented here highlights the structural lay out and formulation of the medical information model. The aim of presenting this to aid medical departments as well as workers to exchange information and integrate available resources that help facilitate the analysis to be conducted on the given information. Software here comprises of medical information and offers a comprehensive service structure that benefits medical data centers. VNM or Virtual Network Mapping (VNM) essentially relates to substrate network that involves the installation and structuring of on demand virtual machines. These however are subjective to certain limitations that are applicable in relation to latency, capacity as well as bandwidth. Data centers need to dynamically handle cloud workloads effectively and efficiently. Simultaneously, since the mapping of virtual and physical networks with several providers’ consumes more time along with energy. In order to resolve this issue, VNM has been mapped by making use of Graph Theory (GT) matching, a well-studied database topic. (i) Brain Storm Optimization Graph Theory (BSOGT) is introduced for modeling a virtual network request in the form of a GT with different resource constraints, and the substrate networks here is considered being a graph. For this graph the nodes and edges comprise of attributes that indicate their constraints. (ii) The algorithm that has been recently introduced executes graph decomposition into several topology patterns. Thereafter the BSOGT is executed to solve any issues that pertain to mapping. (iii) The model that has been presented here, ERVNM and the BSOGT are used with a specific mapping energy computation function.(iv) Issues pertaining to these are categorized as being those related to virtual network mapping as the ACGT and optimal solution are drawn by using effective integer linear programming. ACGT, pragmatic approach, as well as the precise and two-stage algorithms performance is evaluated by means of cloud Simulator environment. The results obtained from simulation indicate that the BSOGT algorithm attains the objectives of cloud service providers with respect to Acceptance ratio, mapping percentage, processing time as well as Convergence Time.
关键词： Virtual Network Mapping (VNM),Distributed cloud computing and optimization,Virtualization quality of services (Qos),Brain Storm Optimization Graph Theory (BSOGT)
更新于2025-09-23 15:22:29
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Specialty probes give super-res imaging that special blink
摘要： Fluorescent probes light the way to cellular detail, but light can also get in the way. Because of the diffraction limit, structures closer to one another than 200 nanometers (nm) or so cannot be discerned. Unless you use probes with super-resolution imaging. These techniques, such as reversible saturable optical linear fluorescence transitions (RESOLFT) or photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM), use specialty probes, dyes and fluorescent proteins (FPs) that can switch from dark to light and from one color to another. 'We need the labels in combination with the microscope to overcome the diffraction barrier,' says Stefan Jakobs, who develops probes at University Medical Center G?ttingen and the Max Planck Institute for Biophysical Chemistry. In structured illumination microscopy (SIM), labs routinely achieve 100-nm resolution, he says. Scientists using stimulated emission depletion microscopy (STED), RESOLFT, PALM or STORM reach beyond 50-nm resolution. In principle, he says, the methods are diffraction unlimited.
关键词： STORM,STED,RESOLFT,super-resolution imaging,SIM,fluorescent probes,diffraction limit,PALM
更新于2025-09-23 15:21:21
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[IEEE IGARSS 2018 - 2018 IEEE International Geoscience and Remote Sensing Symposium - Valencia, Spain (2018.7.22-2018.7.27)] IGARSS 2018 - 2018 IEEE International Geoscience and Remote Sensing Symposium - Improving Wind Forcing with Scatterometer Observations for Operational Storm Surge Forecasting in the Adriatic Sea
摘要： Reliable storm surge predictions rely on accurate atmospheric model simulations, especially of the sea surface pressure and wind vector. The Adriatic Sea is among the regional seas of the Mediterranean basin experiencing the highest tidal excursions, particularly in its northern side, the Gulf of Venice, where storm surge predictions are therefore of great importance. Unfortunately, sea surface wind forecasts in the Adriatic Sea are known to be underestimated. A numerical method aiming at reducing the bias between scatterometer wind observations and atmospheric model winds, has been developed. The method is called “wind bias mitigation” and uses the scatterometer observations to reduce the bias between scatterometer observations and the modeled sea surface wind, in this case that supplied by the European Centre for Medium-Range Weather Forecasts (ECMWF) global atmospheric model. We have compared four mathematical approaches to this method, for a total of eight different formulations of the multiplicative factor ?ws which compensates the model wind underestimation, thus decreasing the bias between scatterometer and model. Four datasets are used for the assessment of the eight different bias mitigation methods: a collection of 29 storm surge events (SEVs) cases in the years 2004-2014, a collection of 48 SEVs in the years 2013-2016, a collection of 364 cases of random sea level conditions in the same period, and a collection of the seven SEVs in 2012-2016 that were worst predicted. The statistical analysis shows that the bias mitigation procedures supplies a mean wind speed more accurate than the standard forecast, when compared with scatterometer observations, in more than 70% of the analyzed cases.
关键词： Sea surface wind,Atmospheric model,Forecasting,Adriatic Sea,Scatterometer,Storm surge
更新于2025-09-23 15:21:21
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Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium <em>Prochlorococcus</em>
摘要： Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, however, the lateral resolution of super-resolution imaging is only ~100 nm. The low resolution is mainly due to the high autofluorescence background of photosynthetic cells caused by high-intensity lasers that are required for super-resolution imaging, such as stochastic optical reconstruction microscopy (STORM). Here, we describe a photobleaching-assisted STORM method which was developed recently for imaging the marine picocyanobacterium Prochlorococcus. After photobleaching, the autofluorescence of Prochlorococcus is effectively reduced so that STORM can be performed with a lateral resolution of ~10 nm. Using this method, we acquire the in vivo three-dimensional (3-D) organization of the FtsZ protein and characterize four different FtsZ ring morphologies during the cell cycle of Prochlorococcus. The method we describe here might be adopted for the super-resolution imaging of other photosynthetic organisms.
关键词： Prochlorococcus,photobleaching,FtsZ ring,Immunology and Infection,STORM,cell division,cyanobacterium,super-resolution imaging,three-dimensional,Issue 141
更新于2025-09-23 15:21:01
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On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores
摘要： While buffer cocktails remain the most commonly used method for photostabilization and photoswitching of fluorescent markers, intramolecular triplet-state quenchers emerge as an alternative strategy to impart fluorophores with 'self-healing' or even functional properties such as photoswitching. In this contribution, we evaluated combinations of both approaches and show that inter- and intramolecular triplet-state quenching processes compete with each other. We find that although the rate of triplet-state quenching is additive, the photostability is limited by the faster pathway. Often intramolecular processes dominate the photophysical situation for combinations of covalently-linked and solution-based photostabilizers and photoswitching agents. Furthermore we show that intramolecular photostabilizers can protect fluorophores from reversible off-switching events caused by solution-additives, which was previously misinterpreted as photobleaching. Our studies also provide practical guidance for usage of photostabilizer–dye conjugates for STORM-type super-resolution microscopy permitting the exploitation of their improved photophysics for increased spatio-temporal resolution. Finally, we provide evidence that the biochemical environment, e.g., proximity of aromatic amino-acids such as tryptophan, reduces the photostabilization efficiency of commonly used buffer cocktails. Not only have our results important implications for a deeper mechanistic understanding of self-healing dyes, but they will provide a general framework to select label positions for optimal and reproducible photostability or photoswitching kinetics in different biochemical environments.
关键词： photoswitching,intramolecular quenching,self-healing dyes,super-resolution microscopy,intermolecular quenching,photostabilization,fluorophores,triplet-state quenching,STORM
更新于2025-09-19 17:15:36
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Localization microscopy at doubled precision with patterned illumination
摘要： MINFLUX offers a breakthrough in single molecule localization precision, but is limited in field of view. Here we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micrometer-sized field of view. We show a near two-fold improvement in precision over standard localization with the same photon count on DNA-origami nanostructures and tubulin in cells, using DNA-PAINT and STORM imaging.
关键词： DNA-origami,DNA-PAINT,patterned illumination,tubulin,STORM,Localization microscopy
更新于2025-09-12 10:27:22
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Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT
摘要： Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore’s response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as “frequency multiplexed DNA-PAINT,” or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.
关键词： STORM,frequency multiplexing,multicolor imaging,DNA-PAINT,superresolution microscopy
更新于2025-09-09 09:28:46
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Self-healing dyes for super-resolution fluorescence microscopy
摘要： In recent years, optical microscopy techniques have emerged that allow optical imaging at unprecedented resolution beyond the diffraction limit. These techniques exploit photostabilizing buffers to enable photoswitching and/or the enhancement of fluorophore brightness and stability. A major drawback with the use of photostabilizing buffers, however, is that they cannot be used in live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores, which undergo intramolecular photostabilization, in super-resolution microscopy examining both targeted (stimulated emission depletion (STED) microscopy) and stochastic readout (stochastic optical reconstruction microscopy (STORM)). The overall goal of the study was to identify dyes and conditions that lead to improved spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. As a result of previously shown superior performance, we identified an ATTO647N-photostabilizer conjugate as a potential candidate for STED microscopy. We have here characterized the photostability and resulting performance of this nitrophenylalanine (NPA) conjugate of ATTO647N on oligonucleotides in STED microscopy. We found that the superior photophysical performance resulted in optimal STED imaging and demonstrated that single-molecule fluorescent transients of individual fluorophores can be obtained with both the excitation- and STED-laser. In similar experiments, we also tested a nitrophenylacetic acid conjugate of STAR635P, another frequently used dye in STED microscopy, and present a characterization of its photophysical properties. Finally, we performed an analysis of the photoswitching kinetics of self-healing Cy5 dyes (containing trolox, cyclooctatetraene and NPA-based stabilizers) in the presence of Tris(2-carboxyethyl) phosphine and cysteamine, which are typically used in STORM microscopy. In line with previous work, we found that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities.
关键词： STORM,super-resolution microscopy,fluorescent dyes,STED,fluorescence microscopy
更新于2025-09-09 09:28:46
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A synthetic review of remote sensing applications to detect nearshore bars
摘要： Nearshore bars are important morphologic features associated with intermediate and dissipative natural beaches. Bars impact the direction, magnitude, and patterns of sediment transport in the nearshore. They serve as a buffer against extreme and meso-scale events. In this review article, we investigate remotely-based observations, specifically near-Earth and satellite imagery, which have been used to investigate nearshore bars. Several recent advances in technology and techniques allow the remote measurement of bar width and height, beach slope, shoreline orientation, and bar count. Video monitoring imagery is presently the most popular method to derive these data. However, spatial prediction models using satellite imagery can also provide reliable bar morphodynamic information.
关键词： Bar monitoring,Coastal morphodynamics,Remote sensing,Geomorphology,Bar storm response
更新于2025-09-04 15:30:14