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[Methods in Molecular Biology] T-Cell Motility Volume 1930 (Methods and Protocols) || Three-Dimensional Structured Illumination Microscopy (3D-SIM) to Dissect Signaling Cross-Talks in Motile T-Cells
摘要： Visualization of signal transduction events in T-cells has always been a challenge due to their miniscule size. Recent advancement in super-resolution microscopy techniques presents many new opportunities to navigate the spatial and temporal signaling cross-talks in motile T-cells. Here, we provide technical details, optimal conditions, and critical practical considerations that need to be taken into account during cell handling, sample preparation, and image acquisition of motile T-cells for performing three-dimensional structured illumination microscopy (3D-SIM).
关键词： 3D-SIM,Immunostaining,Super-resolution microscopy
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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[Lecture Notes in Electrical Engineering] Microelectronics, Electromagnetics and Telecommunications Volume 521 (Proceedings of the Fourth ICMEET 2018) || FSO-Based 5G Mobile Cellular Systems for Urban Canyons
摘要： Over the last few years, there has been a rapid increase in the number of people who are using high data rate services and applications. To be able to support the ever-increasing data rates to the end users, the time has come to consider other technologies which use the upper parts of electromagnetic spectrum other than the Radio Frequency (RF) spectrum. One such technology which can be used as an alternative to RF is Free-Space optical (FSO) communications. It provides higher bandwidth and improved Bit Error Rate (BER) performance because of negligible multipath and fading effect. In this article, the performance of an RF system and an FSO system using BPSK-subcarrier intensity modulation (BPSK-SIM) in a weak turbulent atmosphere are compared. It is found that from simulation results, FSO system has a performance gain of 62% over RF for achieving a BER of 10?5.
关键词： OWC,BER,FSO,BPSK-SIM
更新于2025-09-23 15:21:21
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[Methods in Molecular Biology] Barley Volume 1900 (Methods and Protocols) || Preparation of Barley Pollen Mother Cells for Confocal and Super Resolution Microscopy
摘要： Recombination (crossover) drives the release of genetic diversity in plant breeding programs. However, in barley, recombination is skewed toward the telomeric ends of its seven chromosomes, restricting the re-assortment of about 30% of the genes located in the centromeric regions of its large 5.1 Gb genome. A better understanding of meiosis and recombination could provide ways of modulating crossover distribution and frequency in barley as well as in other grasses, including wheat. While most research on recombination has been carried out in the model plant Arabidopsis thaliana, recent studies in barley (Hordeum Vulgare) have provided new insights into the control of crossing over in large genome species. A major achievement in these studies has been the use of cytological procedures to follow meiotic events. This protocol provides detailed practical steps required to perform immunostaining of barley meiocytes (pollen mother cells) for confocal or structured illumination microscopy.
关键词： Antibodies,Barley,3D-SIM,Immuno-cytochemistry,Confocal Microscopy,Meiosis
更新于2025-09-23 15:21:21
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Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly
摘要： The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro. Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that increasing surface area of the expanding aggresome increases the rate of accretion due to diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.
关键词： molecular modelling,protein aggregation,molecular imaging,passive transport,amyloid protein,aggresome formation,transport,live cell SIM,protein misfolding,molecular dynamics
更新于2025-09-23 15:21:21
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Ionic Current Rectification by Laminated Bipolar Silica Isoporous Membrane
摘要： Ionic current rectification (ICR) is one of interesting characteristics displayed by nanochannels with asymmetric ge-ometry, ionic concentration or charge distribution, which has been utilized for the development of chemical sensors and biosensors. Herein we report the ICR phenomenon observed with ultrathin silica isoporous membrane (SIM), which was prepared by laminat-ing two layers of SIM with opposite charges and different pore dimeters, designated as bipolar SIM (bp-SIM). The negatively charged layer, called as n-SIM, was 86-nm-thick and consisted of channels with a diameter of 2–3 nm. The positively charged layer with a thickness of 59 nm, termed as p-SIM, was comprised of channels of 4.5–5.5 nm in diameter. They were primarily grown on the solid surface using the St?ber-solution and biphasic-stratification growth approaches, respectively, and then exfoliated to obtain perforated structures by the polymer-protected chemical etching and transfer method. The negative charges of n-SIM and positive ones of p-SIM were generated by the deprotonation of pristine surface silanol and post-modified ammonium groups, respectively. Neither n-SIM nor p-SIM alone displays the ICR characteristic, because of their symmetric structure and uniform charge distribu-tion. When laminating two of them, an apparent ICR characteristic was observed for the bp-SIM with a typical diode-like current-voltage response. This behavior was rationalized to arise from the asymmetric charge distribution on two layers by finite element simulations. Considering the facile preparation and diverse surface functionalities, as well as its uniform and highly porous structure, the bp-SIM provides an attractive platform for designing ICR-based sensors.
关键词： bipolar SIM,nanochannels,asymmetric charge distribution,Ionic current rectification,silica isoporous membrane
更新于2025-09-04 15:30:14