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live cell single molecule imaging

Anna-Karin Gustavsson, Petar N Petrov, Maurice Y Lee, Yoav Shechtman, W E Moerner
Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with 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. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts...
January 9, 2018: Nature Communications
Kenichi G N Suzuki, Hiromune Ando, Naoko Komura, Miku Konishi, Akihiro Imamura, Hideharu Ishida, Makoto Kiso, Takahiro K Fujiwara, Akihiro Kusumi
Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking...
2018: Methods in Enzymology
Lan Liu, Jin-Wen Liu, Han Wu, Xiang-Nan Wang, Ru-Qin Yu, Jian-Hui Jiang
Hybridization chain reaction (HCR) circuits are valuable approaches to monitor low-abundance mRNA, and current HCR is still subjected to issues such as limited amplification efficiency, compromised localization resolution or complicated designs. We report a novel branched HCR (bHCR) circuit for efficient signal-amplified imaging of mRNA in living cells. The bHCR can be realized using a simplified design by hierarchically coupling two HCR circuits with two split initiator fragments of the secondary HCR circuit incorporated in the probes for the primary HCR circuit...
January 4, 2018: Analytical Chemistry
Jing Li, Jin Huang, Xiaohai Yang, Yanjing Yang, Ke Quan, Nuli Xie, Yanan Wu, Changbei Ma, Kemin Wang
MicroRNAs (miRNAs) have become an ideal biomarker candidate for early diagnosis of diseases. But various diseases involve changes in the expression of different miRNAs. Therefore, multiplexed assay of miRNAs in live cells can provide critical information for our better understanding of their roles in cells and further validating of their function in clinical diagnoses. Simultaneous detection of multiple biomarkers could effectively improve the accuracy of early cancer diagnosis. Here, we develop the two-color-based nanoflares for simultaneously detecting two distinct miRNA targets inside live cells...
2018: Nanotheranostics
Hui Liu, Peng Dong, Maria S Ioannou, Li Li, Jamien Shea, H Amalia Pasolli, Jonathan B Grimm, Patricia K Rivlin, Luke D Lavis, Minoru Koyama, Zhe Liu
Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic range of ∼10,000-fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by two orders of magnitude...
December 28, 2017: Proceedings of the National Academy of Sciences of the United States of America
Franka Voigt, Hui Zhang, Xianying A Cui, Désirée Triebold, Ai Xin Liu, Jan Eglinger, Eliza S Lee, Jeffrey A Chao, Alexander F Palazzo
It is well established that mRNAs encoding secretory or membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). The extent to which mRNAs that encode cytosolic proteins associate with the ER, however, remains controversial. To address this question, we quantified the number of cytosolic protein-encoding mRNAs that co-localize with the ER using single-molecule RNA imaging in fixed and living cells. We found that a small but significant number of mRNAs that encode cytosolic proteins associate with the ER and show that this interaction is translation dependent...
December 26, 2017: Cell Reports
Stephen J Briddon, Laura E Kilpatrick, Stephen J Hill
G protein-coupled receptors (GPCRs) are organised within the cell membrane into highly ordered macromolecular complexes along with other receptors and signalling proteins. Understanding how heterogeneity in these complexes affects the pharmacology and functional response of these receptors is crucial for developing new and more selective ligands. Fluorescence correlation spectroscopy (FCS) and related techniques such as photon counting histogram (PCH) analysis and image-based FCS can be used to interrogate the properties of GPCRs in these membrane microdomains, as well as their interaction with fluorescent ligands...
December 22, 2017: Trends in Pharmacological Sciences
Anna Lippert, Agnieszka A Janeczek, Alexandre Fürstenberg, Aleks Ponjavic, W E Moerner, Roel Nusse, Jill A Helms, Nicholas D Evans, Steven F Lee
Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding...
December 19, 2017: Biophysical Journal
Minhyeok Chang, Jungsic Oh, Yeonghoon Kim, Sungchul Hohng, Jong-Bong Lee
Real-time optical imaging combined with single-molecule manipulation broadens the horizons for acquiring information about the spatiotemporal localization and the mechanical details of target molecules. To obtain an optical signal outside the focal plane without unintended interruption of the force signal in single-molecule optical imaging-force spectroscopy, we developed an optical method to extend the depth of field in a high numerical aperture objective (≥ 1.2), required to visualize a single fluorophore...
December 11, 2017: Optics Express
Kenji Okamoto, Michio Hiroshima, Yasushi Sako
Single-molecule imaging (SMI) of proteins in operation has a history of intensive investigations over 20 years and is now widely used in various fields of biology and biotechnology. We review the recent advances in SMI of fluorescently-tagged proteins in structural biology, focusing on technical applicability of SMI to the measurements in living cells. Basic technologies and recent applications of SMI in structural biology are introduced. Distinct from other methods in structural biology, SMI directly observes single molecules and single-molecule events one-by-one, thus, explicitly analyzing the distribution of protein structures and the history of protein dynamics...
December 14, 2017: Biophysical Reviews
Rhonda J Davey, Michelle A Digman, Enrico Gratton, Pierre D J Moens
Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A)...
December 11, 2017: Methods: a Companion to Methods in Enzymology
Matthew McDonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Völkl, Michael Aigner, Andreas Mackensen, Vahid Sandoghdar
Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT), and is demonstrated with Laz388 cells-an Epstein-Barr virus (EBV) transformed B cell line...
December 11, 2017: Nano Letters
Amicia D Elliott, Noah Bedard, Alessandro Ustione, Michelle A Baird, Michael W Davidson, Tomasz Tkaczyk, David W Piston
Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges...
2017: PloS One
Harshad Ghodke, Han Ho, Antoine M van Oijen
Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment...
December 1, 2017: Biochemical Society Transactions
Yuki M Shirai, Taka A Tsunoyama, Nao Hiramoto-Yamaki, Koichiro M Hirosawa, Akihiro C E Shibata, Kenichi Kondo, Atsushi Tsurumune, Fumiyoshi Ishidate, Akihiro Kusumi, Takahiro K Fujiwara
Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named "actin-pl-clusters"...
2017: PloS One
Gang Li, Jeffrey E Montgomery, Mark A Eckert, Jae Won Chang, Samantha M Tienda, Ernst Lengyel, Raymond E Moellering
Integration of chemical probes into proteomic workflows enables the interrogation of protein activity, rather than abundance. Current methods limit the biological contexts that can be addressed due to sample homogenization, signal-averaging, and bias toward abundant proteins. Here we report a platform that integrates family-wide chemical probes with proximity-dependent oligonucleotide amplification and imaging to quantify enzyme activity in native contexts with high spatial resolution. Application of this method, activity-dependent proximity ligation (ADPL), to serine hydrolase and cysteine protease enzymes enables quantification of differential enzyme activity resulting from endogenous changes in localization and expression...
November 24, 2017: Nature Communications
Yang Liu, Kornelia Galior, Victor Pui-Yan Ma, Khalid Salaita
Mechanical forces are essential for a variety of biological processes ranging from transcription and translation to cell adhesion, migration, and differentiation. Through the activation of mechanosensitive signaling pathways, cells sense and respond to physical stimuli from the surrounding environment, a process widely known as mechanotransduction. At the cell membrane, many signaling receptors, such as integrins, cadherins and T- or B-cell receptors, bind to their ligands on the surface of adjacent cells or the extracellular matrix (ECM) to mediate mechanotransduction...
November 21, 2017: Accounts of Chemical Research
Wei Wang, Xiaoshan Hou, Xin Li, Chen Chen, Xiliang Luo
In this work, an effective controlled-release biosensor based on Au nanocages (AuNCs) capped with disulfide-containing DNA molecular gates was developed for ultra-sensitive and highly selective detection of glutathione (GSH). Oligonucleotides containing the S-S bonds were assembled on the surface of the AuNCs by means of electrostatic interactions in order to inhibit the release of fluorescent molecules such as Rhodamine B (RhB) loaded by AuNCs. In the presence of GSH, due to the specific cleavage of S-S bonds in disulfide-containing single-stranded DNAs (ssDNAs) as well as their subsequent departure from the surface of AuNCs, the pores could be opened, and then the dye molecules would be released from AuNCs...
January 15, 2018: Analytica Chimica Acta
Nina G Bozhanova, Mikhail S Baranov, Natalia V Klementieva, Karen S Sarkisyan, Alexey S Gavrikov, Ilia V Yampolsky, Elena V Zagaynova, Sergey A Lukyanov, Konstantin A Lukyanov, Alexander S Mishin
We present protein-PAINT - the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes...
October 1, 2017: Chemical Science
Zhenghong Gao, Noémie Danné, Antoine Guillaume Godin, Brahim Lounis, Laurent Cognet
Fluorescence imaging of biological systems down to the single-molecule level has generated many advances in cellular biology. For applications within intact tissue, single-walled carbon nanotubes (SWCNTs) are emerging as distinctive single-molecule nanoprobes, due to their near-infrared photoluminescence properties. For this, SWCNT surfaces must be coated using adequate molecular moieties. Yet, the choice of the suspension agent is critical since it influences both the chemical and emission properties of the SWCNTs within their environment...
November 16, 2017: Nanomaterials
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