Read by QxMD icon Read

live cell single molecule imaging

Thomas Orré, Amine Mehidi, Sophie Massou, Olivier Rossier, Grégory Giannone
To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.
2018: Methods in Molecular Biology
Rajeev Yadav, H Peter Lu
The N-methyl-d-aspartate (NMDA) receptor ion-channel is activated by the binding of ligands, along with the application of action potential, important for synaptic transmission and memory functions. Despite substantial knowledge of the structure and function, the gating mechanism of the NMDA receptor ion channel for electric on-off signals is still a topic of debate. We investigate the NMDA receptor partition distribution and the associated channel's open-close electric signal trajectories using a combined approach of correlating single-molecule fluorescence photo-bleaching, single-molecule super-resolution imaging, and single-channel electric patch-clamp recording...
March 8, 2018: Physical Chemistry Chemical Physics: PCCP
Mingliang Zhang, Zhen Zhang, Kangmin He, Jimin Wu, Nan Li, Rong Zhao, Jinghe Yuan, Han Xiao, Youyi Zhang, Xiaohong Fang
The dynamics and stoichiometry of receptors newly delivered on plasma membrane play a vital role in cell signal transduction, yet the knowledge on this process is limited due to the lack of suitable methods. Here we developed a new strategy, which combines single-molecule imaging (SMI) and fluorescence recovery after photobleaching (FRAP), named FRAP-SMI, to monitor and quantify the newly delivered/inserted single transmembrane receptor on the plasma membrane of living cells. Transforming growth factor-β type II receptor (TβRII), a typical serine/threonine kinase receptor, was studied with this method...
March 6, 2018: Analytical Chemistry
Yanmei Si, Yaocai Bai, Xiaojie Qin, Jun Li, Wenwan Zhong, Zhijun Xiao, Jishan Li, Yadong Yin
A novel ratiometric surface-enhanced Raman scattering (SERS) nanosensor has been developed to probe the activity of endonuclease under in vitro and in living cells conditions. The optimized alloyed Au/Ag nanoparticles (Au/AgNPs) were synthesized as the SERS substrate, which combined the superior properties of both the pure Au and Ag nanoparticles: they exhibit excellent plasmonic property with high chemical stability and low cytotoxicity. They were then employed for quantitative detection of endonuclease through functionalization with single-stranded DNA (ssDNA) carrying 3-(4-(phenylethynyl)benzylthio) propanoic acid (PEB) as endonuclease-responsive SERS signaling molecule, and 4-thiol phenylacetylene (TPA) as the internal standard SERS signaling molecule...
March 5, 2018: Analytical Chemistry
Chen Chen, Shenfei Zong, Zhuyuan Wang, Ju Lu, Dan Zhu, Yizhi Zhang, Ruohu Zhang, Yiping Cui
Exosomes are small membrane vesicles secreted by a wide variety of cells. Studies have demonstrated that exosomal miRNAs can influence the biological processes of recipient cells. Therefore, direct imaging and tracking of exosomal miRNAs in living recipient cells are essential for exosome functional analysis. However, the moderate spatial resolution of conventional fluorescence microscopy limits the precise imaging and tracking of exosomes considering their relatively small size (<100 nm). Here, we took advantage of single molecule localization microscopy (SMLM) to realize the visualization and dynamic tracking of exosomes and exosomal miRNAs in living cells...
March 1, 2018: Nanoscale
Li Wang, Yiqun Xue, Jingjing Xing, Kai Song, Jinxing Lin
Plasma membrane proteins have important roles in transport and signal transduction. Deciphering the spatiotemporal organization of these proteins provides crucial information for elucidating the links between the behaviors of different molecules. However, monitoring membrane proteins without disrupting their membrane environment remains difficult. Over the past decade, many studies have developed single-molecule techniques, opening avenues for probing the stoichiometry and interactions of membrane proteins in their native environment by providing nanometer-scale spatial information and nanosecond-scale temporal information...
February 28, 2018: Annual Review of Plant Biology
Ayuko Sakane, Shin Yoshizawa, Hideo Yokota, Takuya Sasaki
Collective cell migration is observed during morphogenesis, angiogenesis, and wound healing, and this type of cell migration also contributes to efficient metastasis in some kinds of cancers. Because collectively migrating cells are much better organized than a random assemblage of individual cells, there seems to be a kind of order in migrating clusters. Extensive research has identified a large number of molecules involved in collective cell migration, and these factors have been analyzed using dramatic advances in imaging technology...
2018: Frontiers in Cell and Developmental Biology
Yair Razvag, Yair Neve-Oz, Julia Sajman, Meital Reches, Eilon Sherman
T cells have a central function in mounting immune responses. However, mechanisms of their early activation by cognate antigens remain incompletely understood. Here we use live-cell multi-colour single-molecule localization microscopy to study the dynamic separation between TCRs and CD45 glycoprotein phosphatases in early cell contacts under TCR-activating and non-activating conditions. Using atomic force microscopy, we identify these cell contacts with engaged microvilli and characterize their morphology, rigidity and dynamics...
February 21, 2018: Nature Communications
Karl Gatterdam, Eike F Joest, Marina S Dietz, Mike Heilemann, Robert Tampé
Live-cell labeling, super-resolution microscopy, single-molecule applications, protein localization or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Here, we define a new class of hexavalent N-nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA-based small interaction pairs described so far...
February 20, 2018: Angewandte Chemie
Benedikt Niewidok, Maxim Igaev, Abel Pereira da Graca, Andre Strassner, Christine Lenzen, Christian P Richter, Jacob Piehler, Rainer Kurre, Roland Brandt
Stress granules (SGs) are cytosolic, nonmembranous RNA-protein complexes. In vitro experiments suggested that they are formed by liquid-liquid phase separation; however, their properties in mammalian cells remain unclear. We analyzed the distribution and dynamics of two paradigmatic RNA-binding proteins (RBPs), Ras GTPase-activating protein SH3-domain-binding protein (G3BP1) and insulin-like growth factor II mRNA-binding protein 1 (IMP1), with single-molecule resolution in living neuronal cells. Both RBPs exhibited different exchange kinetics between SGs...
February 20, 2018: Journal of Cell Biology
Rui Yan, Seonah Moon, Samuel J Kenny, Ke Xu
As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples...
February 14, 2018: Accounts of Chemical Research
Diana Di Paolo, Richard M Berry
For the last 2 decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signaling in E. coli, including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical. For example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart. FPs may interfere with the native interactions of the protein, and their chromophores have low brightness and photostability, and fast photobleaching rates...
2018: Methods in Molecular Biology
Xuehui Pang, Cheng Cui, Shuo Wan, Ying Jiang, Liangliang Zhang, Lian Xia, Long Li, Xiaowei Li, Weihong Tan
Currently, functional single-stranded oligonucleotide probes, termed aptamers, generated by an iterative technology, Systematic Evolution of Ligands by Exponential Enrichment (SELEX), are utilized to selectively target molecules or cells with high affinity. Aptamers hold considerable promise as multifunctional molecules or conjugates for challenging nanotechnologies or bioapplications now and in the future. In this review, we first describe recent endeavors to select aptamers towards live cancer cells via cell-SELEX...
February 9, 2018: Cancers
Waldemar Schrimpf, Veerle Lemmens, Nick Smisdom, Marcel Ameloot, Don C Lamb, Jelle Hendrix
Raster image cross-correlation spectroscopy (ccRICS) can be used to quantify the interaction affinities between diffusing molecules by analyzing the fluctuations between two-color confocal images. Spectral crosstalk compromises the quantitative analysis of ccRICS experiments, limiting multicolor implementations to dyes with well-separated emission spectra. Here, we remove this restriction by introducing raster spectral image correlation spectroscopy (RSICS), which employs statistical filtering based on spectral information to quantitatively separate signals of fluorophores during spatial correlation analysis...
February 2, 2018: Methods: a Companion to Methods in Enzymology
Tao Huang, Carey Phelps, Jing Wang, Li-Jung Lin, Amy Bittel, Zubenelgenubi Scott, Steven Jacques, Summer L Gibbs, Joe W Gray, Xiaolin Nan
Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels...
January 23, 2018: Biophysical Journal
Zhe Liu, Robert Tjian
The assembly of sequence-specific enhancer-binding transcription factors (TFs) at cis-regulatory elements in the genome has long been regarded as the fundamental mechanism driving cell type-specific gene expression. However, despite extensive biochemical, genetic, and genomic studies in the past three decades, our understanding of molecular mechanisms underlying enhancer-mediated gene regulation remains incomplete. Recent advances in imaging technologies now enable direct visualization of TF-driven regulatory events and transcriptional activities at the single-cell, single-molecule level...
January 29, 2018: Journal of Cell Biology
Tai-Yen Chen, Yu-Shan Cheng, Pei-San Huang, Peng Chen
Dynamic protein-DNA interactions constitute highly robust cellular machineries to fulfill cellular functions. A vast number of studies have focused on how DNA-binding proteins search for and interact with their target DNA segments and on what cellular cues can regulate protein binding, for which protein concentration is a most obvious one. In contrast, how protein unbinding could be regulated by protein concentration has evaded attention because protein unbinding from DNA is typically a unimolecular reaction and thus concentration independent...
January 25, 2018: Accounts of Chemical Research
Adekunle T Bademosi, Elsa Lauwers, Rumelo Amor, Patrik Verstreken, Bruno van Swinderen, Frédéric A Meunier
An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane...
January 14, 2018: Journal of Visualized Experiments: JoVE
Leonel Malacrida, Per Niklas Hedde, Suman Ranjit, Francesco Cardarelli, Enrico Gratton
Despite recent advances in optical super-resolution, we lack a method that can visualize the path followed by diffusing molecules in the cytoplasm or in the nucleus of cells. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level by averaging the behavior of many molecules over time at a single spot, thus achieving very good statistics but at only one point in the cell. Earlier image-based methods including raster-scan and spatiotemporal image correlation need spatial averaging over relatively large areas, thus compromising spatial resolution...
January 1, 2018: Biomedical Optics Express
Evelina Tutucci, Nathan M Livingston, Robert H Singer, Bin Wu
RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred fromDNAto proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution...
January 18, 2018: Annual Review of Biophysics
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"