Browse
Submit Data
Databases
API
Help

Dataset Information

60 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Photoregulated fluxional fluorophores for live-cell super-resolution microscopy with no apparent photobleaching.

ABSTRACT: Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30?min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.

SUBMITTER: Halabi EA

PROVIDER: S-EPMC6420572 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Photoregulated fluxional fluorophores for live-cell super-resolution microscopy with no apparent photobleaching.

Halabi Elias A EA Pinotsi Dorothea D Rivera-Fuentes Pablo P

Nature communications 20190315 1

Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables ...[more]

PMID: 30874551

Similar Datasets

Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores.

Project description:Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. While, in the case of SIM (upon epi-illumination), cell layers of about 1-2 µm in close proximity to the plasma membrane can be selected by software, layers in the 100 nm range are assessed experimentally by TIRF-SIM. To show the applicability of this approach, both methods are used to measure the translocation of the glucose transporter 4 (GLUT4) from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds, with a lateral resolution of around 100 nm and an axial resolution of around 200 nm. While SIM is an appropriate method to visualize the intracellular localization of GLUT4 fused with a green fluorescent protein, TIRF-SIM permits the quantitative evaluation of its fluorescence in the plasma membrane. These imaging methods are discussed in the context of fluorescence lifetime kinetics, providing additional data for the molecular microenvironment.

| S-EPMC7582769 | biostudies-literature

Light-induced cell damage in live-cell super-resolution microscopy.

Project description:Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24?hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1?kW cm(-2) at 640?nm for several minutes, the maximum dose at 405?nm is only ~50?J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.

| S-EPMC4611486 | biostudies-literature

A microfluidic platform for correlative live-cell and super-resolution microscopy.

Project description:Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.

| S-EPMC4278722 | biostudies-literature

Super-beacons: Open-source probes with spontaneous tuneable blinking compatible with live-cell super-resolution microscopy.

Project description:Localization-based super-resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non-fluorescent states. These transitions are commonly regulated by high-intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single-molecule self-quenching as a mechanism to generate spontaneous photoswitching. To demonstrate this principle, we developed a new class of DNA-based open-source super-resolution probes named super-beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live-cell compatible super-resolution microscopy without high-illumination or toxic imaging buffers is revealed by imaging interferon inducible transmembrane proteins (IFITMs) at sub-100 nm resolutions.

| S-EPMC7643006 | biostudies-literature

Multi-color live-cell super-resolution volume imaging with multi-angle interference microscopy.

Project description:Imaging and tracking of near-surface three-dimensional volumetric nanoscale dynamic processes of live cells remains a challenging problem. In this paper, we propose a multi-color live-cell near-surface-volume super-resolution microscopy method that combines total internal reflection fluorescence structured illumination microscopy with multi-angle evanescent light illumination. We demonstrate that our approach of multi-angle interference microscopy is perfectly adapted to studying subcellular dynamics of mitochondria and microtubule architectures during cell migration.

| S-EPMC6240104 | biostudies-literature

Small-molecule labeling of live cell surfaces for three-dimensional super-resolution microscopy.

Project description:Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10-20 nm.

| S-EPMC4195381 | biostudies-literature

Super-resolution video microscopy of live cells by structured illumination.

Project description:Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.

| S-EPMC2895555 | biostudies-literature

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy.

Project description:Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150?nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

| S-EPMC4964612 | biostudies-literature

Graphene-Enabled, Spatially Controlled Electroporation of Adherent Cells for Live-Cell Super-resolution Microscopy.

Project description:The incorporation of exogenous molecules into live cells is essential for both biological research and therapeutic applications. In particular, for the emerging field of super-resolution microscopy of live mammalian cells, it remains a challenge to deliver tailored, often cell-impermeable, fluorescent probes into live cells for target labeling. Here, utilizing the outstanding mechanical, electrical, and optical properties of graphene, we report a facile approach that enables both high-throughput delivery of fluorescent probes into adherent mammalian cells and in situ super-resolution microscopy on the same device. Approximately 90% delivery efficiencies are achieved for free dyes and dye-tagged affinity probes, short peptides, and whole antibodies, thus enabling high-quality super-resolution microscopy. Moreover, we demonstrate good spatiotemporal controls, which, in combination with the ready patternability of graphene, allow for the spatially selective delivery of two different probes for cells at different locations on the same substrate.

| S-EPMC7448575 | biostudies-literature

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy.

Project description:We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.

| S-EPMC6959352 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data