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live cell single molecule imaging

Dongmyung Oh, Yang Yu, Hochan Lee, Jae-Hyung Jeon, Barry L Wanner, Ken Ritchie
The spatial location of proteins in living cells can be critical for their function. For example, the E. coli chemotaxis machinery is localized to the cell poles. Here we describe the polar localization of the serine chemoreceptor Tsr using a strain synthesizing a fluorescent Tsr-Venus fusion at a low level from a single-copy chromosomal construct. Using photobleaching and imaging during recovery by new synthesis, we observed distinct asymmetry between a bright (old) pole and a dim (new) pole. The old pole was shown to be a more stable cluster and to recover after photobleaching faster, which is consistent with the hypothesis that newly synthesized Tsr proteins are inserted directly at or near the old pole...
2018: PloS One
Benoit Roubinet, Matthias Bischoff, Shamil Nizamov, Sergey Yan, Claudia Geisler, Stefan Stoldt, Gyuzel Y Mitronova, Vladimir N Belov, Mariano L Bossi, Stefan W Hell
Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor superresolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-L-alanyl - beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides)...
May 11, 2018: Journal of Organic Chemistry
Aleks Ponjavic, James McColl, Alexander R Carr, Ana Mafalda Santos, Klara Kulenkampff, Anna Lippert, Simon J Davis, David Klenerman, Steven F Lee
Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i...
May 8, 2018: Biophysical Journal
Marcin Poreba, Wioletta Rut, Matej Vizovisek, Katarzyna Groborz, Paulina Kasperkiewicz, Darren Finlay, Kristiina Vuori, Dusan Turk, Boris Turk, Guy S Salvesen, Marcin Drag
Cysteine cathepsins normally function in the lysosomal degradation system where they are critical for the maintenance of cellular homeostasis and the MHC II immune response, and have been found to have major roles in several diseases and in tumor progression. Selective visualization of individual protease activity within a complex proteome is of major importance to establish their roles in both normal and tumor cells, thereby facilitating our understanding of the regulation of proteolytic networks. A generally accepted means to monitor protease activity is the use of small molecule substrates and activity-based probes...
February 28, 2018: Chemical Science
Anna-Karin Gustavsson, Petar N Petrov, Maurice Y Lee, Yoav Shechtman, W E Moerner
To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts...
February 2018: Proceedings of SPIE
Achillefs N Kapanidis, Alessia Lepore, Meriem El Karoui
Bacteria are microorganisms central to health and disease, serving as important model systems for our understanding of molecular mechanisms and for developing new methodologies and vehicles for biotechnology. In the past few years, our understanding of bacterial cell functions has been enhanced substantially by powerful single-molecule imaging techniques. Using single fluorescent molecules as a means of breaking the optical microscopy limit, we can now reach resolutions of ∼20 nm inside single living cells, a spatial domain previously accessible only by electron microscopy...
April 18, 2018: Biophysical Journal
Han N Ho, Antoine M van Oijen, Harshad Ghodke
During transcription elongation, bacterial RNA polymerase (RNAP) can pause, backtrack or stall when transcribing template DNA. Stalled transcription elongation complexes at sites of bulky lesions can be rescued by the transcription terminator Mfd. The molecular mechanisms of Mfd recruitment to transcription complexes in vivo remain to be elucidated, however. Using single-molecule live-cell imaging, we show that Mfd associates with elongation transcription complexes even in the absence of exogenous genotoxic stresses...
April 20, 2018: Nature Communications
Wei Ouyang, Andrey Aristov, Mickaël Lelek, Xian Hao, Christophe Zimmer
The speed of super-resolution microscopy methods based on single-molecule localization, for example, PALM and STORM, is limited by the need to record many thousands of frames with a small number of observed molecules in each. Here, we present ANNA-PALM, a computational strategy that uses artificial neural networks to reconstruct super-resolution views from sparse, rapidly acquired localization images and/or widefield images. Simulations and experimental imaging of microtubules, nuclear pores, and mitochondria show that high-quality, super-resolution images can be reconstructed from up to two orders of magnitude fewer frames than usually needed, without compromising spatial resolution...
April 16, 2018: Nature Biotechnology
Laura Avogaro, Emmanuelle Querido, Myriam Dalachi, Michael F Jantsch, Pascal Chartrand, Emilio Cusanelli
Telomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres...
April 16, 2018: RNA Biology
Eric H Kim, Gregory Chin, Guoxin Rong, Kira E Poskanzer, Heather A Clark
Fluorescent nanosensors and molecular probes are next-generation tools for imaging chemical signaling inside and between cells. Electrophysiology has long been considered the gold standard in elucidating neural dynamics with high temporal resolution and precision, particularly on the single-cell level. However, electrode-based techniques face challenges in illuminating the specific chemicals involved in neural cell activation with adequate spatial information. Measuring chemical dynamics is of fundamental importance to better understand synergistic interactions between neurons as well as interactions between neurons and non-neuronal cells...
April 13, 2018: Accounts of Chemical Research
Yu-Wen Su, Wei Wang
Surface plasmon resonance (SPR) has become a well-recognized label-free technique for measuring the binding kinetics between biomolecules since the invention of the first SPR-based immunosensor in 1980s. The most popular and traditional format for SPR analysis is to monitor the real-time optical signals when a solution containing ligand molecules is flowing over a sensor substrate functionalized with purified receptor molecules. In recent years, rapid development of several kinds of SPR imaging techniques have allowed for mapping the dynamic distribution of local mass density within single living cells with high spatial and temporal resolutions and reliable sensitivity...
April 12, 2018: Analytical and Bioanalytical Chemistry
Marie-Lena I E Harwardt, Marina S Dietz, Mike Heilemann, Thorsten Wohland
The dynamics of biomolecules in the plasma membrane is of fundamental importance to understanding cellular processes. Cellular signaling often starts with extracellular ligand binding to a membrane receptor, which then transduces an intracellular signal. Ligand binding and receptor-complex activation often involve a complex rearrangement of proteins in the membrane, which results in changes in diffusion properties. Two widely used methods to characterize biomolecular diffusion are single-particle tracking (SPT) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS)...
April 9, 2018: Biophysical Journal
Yasaman Ardeshirpour, Dan L Sackett, Jay R Knutson, Amir H Gandjbakhche
BACKGROUND: Assessment of the status of tumor biomarkers in individual patients would facilitate personalizing treatment strategy, and continuous monitoring of those biomarkers and their binding process to the therapeutic drugs would provide a means for early evaluation of the efficacy of therapeutic intervention. Fluorescent probes can accumulate inside the tumor region due to the leakiness of its vascularization and this can make it difficult to distinguish if the measured fluorescence intensity is from probes bound to target receptors or just accumulated unbound probes inside the tumor...
April 4, 2018: EJNMMI Research
Shingo Sotoma, Daiki Terada, Takuya F Segawa, Ryuji Igarashi, Yoshie Harada, Masahiro Shirakawa
The development of sensors to estimate physical properties, and their temporal and spatial variation, has been a central driving force in scientific breakthroughs. In recent years, nanosensors based on quantum measurements, such as nitrogen-vacancy centres (NVCs) in nanodiamonds, have been attracting much attention as ultrastable, sensitive, accurate and versatile physical sensors for quantitative cellular measurements. However, the nanodiamonds currently available for use as sensors have diameters of several tens of nanometres, much larger than the usual size of a protein...
April 3, 2018: Scientific Reports
Taka A Tsunoyama, Yusuke Watanabe, Junri Goto, Kazuma Naito, Rinshi S Kasai, Kenichi G N Suzuki, Takahiro K Fujiwara, Akihiro Kusumi
Single-fluorescent-molecule imaging tracking (SMT) is becoming an important tool to study living cells. However, photobleaching and photoblinking (hereafter referred to as photobleaching/photoblinking) of the probe molecules strongly hamper SMT studies of living cells, making it difficult to observe in vivo molecular events and to evaluate their lifetimes (e.g., off rates). The methods used to suppress photobleaching/photoblinking in vitro are difficult to apply to living cells because of their toxicities. Here using 13 organic fluorophores we found that, by combining low concentrations of dissolved oxygen with a reducing-plus-oxidizing system, photobleaching/photoblinking could be strongly suppressed with only minor effects on cells, which enabled SMT for as long as 12,000 frames (~7 min at video rate, as compared to the general 10-s-order durations) with ~22-nm single-molecule localization precisions...
April 2, 2018: Nature Chemical Biology
Emrah Eroglu, Helmut Bischof, Suphachai Charoensin, Markus Waldeck-Weiermaier, Wolfgang F Graier, Roland Malli
Nitric oxide (NO• ) is a versatile signaling molecule which regulates fundamental cellular processes in all domains of life. However, due to the radical nature of NO• it has a very short half-life that makes it challenging to trace its formation, diffusion, and degradation on the level of individual cells. Very recently, we expanded the family of genetically encoded sensors by introducing a novel class of single fluorescent protein-based NO• probes-the geNOps. Once expressed in cells of interest, geNOps selectively respond to NO• by fluorescence quench, which enables real-time monitoring of cellular NO• signals...
2018: Methods in Molecular Biology
Ryosuke Fukushima, Johtaro Yamamoto, Hideto Ishikawa, Masataka Kinjo
Number and brightness analysis (N&B) is a useful tool for the simultaneous visualization of protein oligomers and their localization, with single-molecule sensitivity. N&B determines particle brightness (fluorescence intensity per particle) and maps the spatial distribution of fluorescently labeled proteins by performing statistical analyses of the image series obtained using laser scanning microscopy. The brightness map reveals presence of the oligomers of the targeted protein and their distribution in living cells...
March 20, 2018: Methods: a Companion to Methods in Enzymology
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 28, 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 the plasma membrane play a vital role in cell signal transduction, yet knowledge of this process is limited because of the lack of suitable analytical methods. Here we developed a new strategy that combines single-molecule imaging (SMI) and fluorescence recovery after photobleaching (FRAP), named FRAP-SMI, to monitor and quantify individual newly delivered and inserted transmembrane receptors on plasma membranes of living cells. Transforming-growth-factor-β type II receptor (TβRII), a typical serine/threoninekinase receptor, was studied with this method...
April 3, 2018: Analytical Chemistry
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