scholarly journals FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

2020 ◽  
Author(s):  
Matthieu Lagardère ◽  
Ingrid Chamma ◽  
Emmanuel Bouilhol ◽  
Macha Nikolski ◽  
Olivier Thoumine
Keyword(s):  
Real Time ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Simulated Data ◽  
Super Resolution ◽  
Molecular Complexes ◽  
Live Cell ◽  
Single Molecule Level ◽  
Resolution Imaging

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive real-time simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexin-neuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.

scholarly journals FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Matthieu Lagardère ◽  
Ingrid Chamma ◽  
Emmanuel Bouilhol ◽  
Macha Nikolski ◽  
Olivier Thoumine
Keyword(s):  
Membrane Protein ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Simulated Data ◽  
Super Resolution ◽  
Molecular Complexes ◽  
Live Cell ◽  
Resolution Imaging ◽  
Membrane Protein Dynamics

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these issues, we present here a robust computer program, called FluoSim, which is an interactive simulator of membrane protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. FluoSim integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the localization and intensity of thousands of independent molecules in 2D cellular geometries, providing simulated data directly comparable to actual experiments. FluoSim was thoroughly validated against experimental data obtained on the canonical neurexin-neuroligin adhesion complex at cell–cell contacts. This unified software allows one to model and predict membrane protein dynamics and localization at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments.

scholarly journals Nanoscopy on the Chea(i)p

2020 ◽  
Author(s):  
Benedict Diederich ◽  
Øystein Helle ◽  
Patrick Then ◽  
Pablo Carravilla ◽  
Kay Oliver Schink ◽  
...  
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Low Cost ◽  
Super Resolution ◽  
Image Sensor ◽  
Optical Diffraction ◽  
Sensor Technology ◽  
Safety Level ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

AbstractSuper-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.

scholarly journals Super-Resolution Imaging of Tight and Adherens Junctions: Challenges and Open Questions

2020 ◽  
Vol 21 (3) ◽  
pp. 744 ◽  
Author(s):  
Hannes Gonschior ◽  
Volker Haucke ◽  
Martin Lehmann
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Rapid Development ◽  
Ion Homeostasis ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Molecular Architecture ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging

The tight junction (TJ) and the adherens junction (AJ) bridge the paracellular cleft of epithelial and endothelial cells. In addition to their role as protective barriers against bacteria and their toxins they maintain ion homeostasis, cell polarity, and mechano-sensing. Their functional loss leads to pathological changes such as tissue inflammation, ion imbalance, and cancer. To better understand the consequences of such malfunctions, the junctional nanoarchitecture is of great importance since it remains so far largely unresolved, mainly because of major difficulties in dynamically imaging these structures at sufficient resolution and with molecular precision. The rapid development of super-resolution imaging techniques ranging from structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (SMLM) has now enabled molecular imaging of biological specimens from cells to tissues with nanometer resolution. Here we summarize these techniques and their application to the dissection of the nanoscale molecular architecture of TJs and AJs. We propose that super-resolution imaging together with advances in genome engineering and functional analyses approaches will create a leap in our understanding of the composition, assembly, and function of TJs and AJs at the nanoscale and, thereby, enable a mechanistic understanding of their dysfunction in disease.

Competitive multicomponent anion exchange adsorption of proteins at the single molecule level

The Analyst ◽  
2017 ◽  
Vol 142 (17) ◽  
pp. 3127-3131 ◽  
Author(s):  
Lydia Kisley ◽  
Ujwal Patil ◽  
Sagar Dhamane ◽  
Katerina Kourentzi ◽  
Lawrence J. Tauzin ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Single Molecule ◽  
Adsorption Kinetics ◽  
Competitive Adsorption ◽  
Super Resolution ◽  
Single Molecule Level ◽  
Exchange Adsorption ◽  
Resolution Imaging

Super-resolution imaging of multicomponent, competitive adsorption demonstrates that competitors block certain ligands from the analyte without changing analyte adsorption kinetics.

scholarly journals Active DNA unwinding and transport by a membrane-adapted helicase nanopore

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ke Sun ◽  
Changjian Zhao ◽  
Xiaojun Zeng ◽  
Yuejia Chen ◽  
Xin Jiang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Single Cell Analysis ◽  
Single Channel ◽  
Vital Role ◽  
Live Cell ◽  
Bovine Papillomavirus ◽  
Nanoscale Transport ◽  
Single Molecule Level

Abstract Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.

scholarly journals Imaging the ultrastructures and dynamics of live erythrocyte membranes at the single-molecule level with a far-red probe on a microfluidic platform

2021 ◽  
Author(s):  
Zhiwei Ye ◽  
Wei Yang ◽  
Shujing Wang ◽  
Ying Zheng ◽  
Xiaodong Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Clinical Diagnostics ◽  
Erythrocyte Membranes ◽  
Single Molecule Tracking ◽  
Microfluidic Platform ◽  
Single Molecule Level ◽  
Diagnostic Approaches

AbstractBoth the ultrastructures and dynamics of living erythrocyte membranes provide critical criteria for clinical diagnostics. However, it is challenging to simultaneously visualize these features at the single-molecule level due to the rigid photophysical requirements of different single-molecule imaging techniques. Herein, we rationally developed a far-red boron dipyrromethene membrane (BDP-Mem) probe that not only retained consistent and intensive single-molecule emission but also possessed the capability to photoswitch on cellular membranes. We also constructed a microfluidic platform for the noninvasive trapping and long-term imaging of nonadherent erythrocytes. By combining these advantageous technologies, super-resolution reconstruction and single-molecule tracking of living human RBC membranes were achieved at the molecular scale in a high-throughput fashion. Our integrated paradigm defines a quantitative approach for analyzing living RBC membranes under physiological and pathological conditions, improving imaging precisions and revealing new perspectives for future disease diagnostic approaches.

scholarly journals Direct Observation of Anisotropy-Driven Formation of Amyloid Protein Core-Shell Structures in Real-time by Super-resolution Microscopy

2021 ◽  
Author(s):  
Min Zhang ◽  
Henrik Dahl Pinholt ◽  
Xin Zhou ◽  
Soeren S-R Bohr ◽  
Luca Banneta ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Growth Rates ◽  
Super Resolution ◽  
Average Growth ◽  
Single Molecule Level ◽  
Aggregation Processes ◽  
Morphological Transitions ◽  
Super Resolution Microscopy ◽  
Kinetics Of

Proteins misfolding and aggregation in the form of fibrils or amyloid containing spherulite-like structures, are involved in a spectrum of degenerative diseases. Current understanding of protein aggregation mechanism primarily relies on conventional spectrometric methods reporting the average growth rates and microscopy readouts of final structures, consequently masking the morphological and growth heterogeneity of the aggregates. Here we developed REal-time kinetics via binding and Photobleaching LOcalization Microscopy (REPLOM) super resolution method to observe directly and quantify the existence and abundance of diverse aggregation morphologies as well as the heterogeneous growth kinetics of each of them. Our results surprisingly revealed insulin aggregation is not exclusively isotropic, but it may also occur anisotropically. Combined with Machine learning we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape for each aggregation morphology. Our unifying framework of detection and analysis of spherulite growth can be extended to other protein systems and reveal their aggregation processes at single molecule level.

scholarly journals A spontaneously blinking fluorescent protein for simple single laser super-resolution live cell imaging

2017 ◽  
Author(s):  
Yoshiyuki Arai ◽  
Hiroki Takauchi ◽  
Yuhei Ogami ◽  
Satsuki Fujiwara ◽  
Masahiro Nakano ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Fluorescent Protein ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Light Illumination ◽  
Excitation Light ◽  
Thermally Induced ◽  
Resolution Imaging ◽  
Single Molecule Localization Microscopy

AbstractSuper-resolution imaging techniques based on single molecule localization microscopy (SMLM) broke the diffraction limit of optical microscopy in living samples with the aid of photoswitchable fluorescent probes and intricate microscopy systems. Here, we developed a fluorescent protein, SPOON, which can be switched-off by excitation light illumination and switched-on by thermally-induced dehydration resulting in an apparent spontaneous blinking behavior. This unique property of SPOON provides a simple SMLM-based super-resolution imaging platform which requires only a single 488 nm laser.

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