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 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 EXPRESS: Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

2021 ◽  
pp. 000370282110099
Author(s):  
Ziyu Yang ◽  
Haiqi Xu ◽  
Jiayu Wang ◽  
Wei Chen ◽  
Meiping Zhao
Keyword(s):  
Membrane Protein ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Membrane Receptors ◽  
Biological Macromolecules ◽  
Dynamics Analysis ◽  
Single Molecule Fluorescence ◽  
Membrane Protein Dynamics

Fluorescence-based single molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labelling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labelling sites, experimental setup and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

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.

Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells

2015 ◽  
Vol 10 (12) ◽  
pp. 2054-2063 ◽  
Author(s):  
Xiaohua Wang ◽  
Xiaojuan Li ◽  
Xin Deng ◽  
Doan-Trung Luu ◽  
Christophe Maurel ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Dynamics ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Plant Cells ◽  
Single Molecule Fluorescence ◽  
Living Plant ◽  
Membrane Protein Dynamics

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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