Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Robust scan synchronized force-fluorescence imaging.

ABSTRACT: Simultaneous atomic force microscope (AFM) and sample scanning confocal fluorescence microscope measurements are widely used to obtain mechanistic and structural insights into protein dynamics in live cells. However, the absence of a robust technique to synchronously scan both AFM and confocal microscope piezo stages makes it difficult to visualize force-induced changes in fluorescent protein distribution in cells. To address this challenge, we have built an integrated AFM-confocal fluorescence microscope platform that implements a synchronous scanning method which eliminates image artifacts from piezo motion ramping, produces accurate pixel binning and enables the collection of a scanned image of a sample while applying force to a single point on the sample. As proof of principle, we use this instrument to monitor the redistribution of fluorescent E-cadherin, an essential transmembrane protein, in live cells, upon application of mechanical force.

SUBMITTER: Schmidt P

PROVIDER: S-EPMC8761069 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging.

Project description:Integrin-transmitted cellular forces are critical for platelet adhesion, activation, aggregation and contraction during hemostasis and thrombosis. Measuring and mapping single platelet forces are desired in both research and clinical applications. Conventional force-to-strain based cell traction force microscopies have low resolution which is not ideal for cellular force mapping in small platelets. To enable platelet force mapping with submicron resolution, we developed a force-activatable biosensor named integrative tension sensor (ITS) which directly converts molecular tensions to fluorescent signals, therefore enabling cellular force mapping directly by fluorescence imaging. With ITS, we mapped cellular forces in single platelets at 0.4µm resolution. We found that platelet force distribution has strong polarization which is sensitive to treatment with the anti-platelet drug tirofiban, suggesting that the ITS force map can report anti-platelet drug efficacy. The ITS also calibrated integrin molecular tensions in platelets and revealed two distinct tension levels: 12-54 piconewton (nominal values) tensions generated during platelet adhesion and tensions above 54 piconewton generated during platelet contraction. Overall, the ITS is a powerful biosensor for the study of platelet mechanobiology, and holds great potential in antithrombotic drug development and assessing platelet activity in health and disease.

| S-EPMC6552678 | biostudies-literature

Radioactivity-synchronized fluorescence enhancement using a radionuclide fluorescence-quenched dye.

Project description:We demonstrate the first evidence of radioactivity-synchronized fluorescence quenching of a near-infrared light-emitting dye by a radionuclide, (64)Cu, and subsequent fluorescence enhancement upon (64)Cu decay to the daughter isotopes (64)Ni and (64)Zn. The dynamic switch from high radioactivity and low fluorescence to low radioactivity and high fluorescence is potentially useful for developing complementary multimodal imaging and detection platforms for chemical, environmental, and biomedical applications as well as for unraveling the mechanisms of metal-induced dynamic fluorescence changes.

| S-EPMC2715832 | biostudies-literature

Precise immuno-fluorescence canceling enables highly multiplexed imaging

Project description:This SuperSeries is composed of the SubSeries listed below.

2024-04-16 | GSE242887 | GEO

Precise immuno-fluorescence canceling enables highly multiplexed imaging [RNA-seq]

Project description:Cell states are regulated by extrinsic signals from various external factors such as intercellular interactions, and intrinsic gene expression. Although comprehensive cell state profiling has been attempted, it remains simultaneous analysis of signal activation has still been challenging. Multiplexed imaging is a technique acquiring multiple protein information at a single cell level as traditional immunofluorescence. However, the method often compromises resolution, hindering the analysis of intracellular localization dynamics and post-translational modifications of proteins. To address these limitations, we developed an erasable fluorescence method using disulfide linkers to label antibodies. We term these antibodies ‘Precise Emission Canceling Antibodies (PECAbs)’. PECAb allows for high-resolution iterative imaging with minimal non-specific binding. Automation enables our system to achieve reproducible quantitative analysis using 206 antibodies. The resulting quantitative data allow reconstruction of the spatiotemporal dynamics of signaling pathways over both long and short timescales. Additionally, combining this approach with sequential RNA-FISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system serves as a comprehensive platform for analyzing complex cell processes, from signal transduction to gene expression.

2024-04-21 | GSE242886 | GEO

Precise immuno-fluorescence canceling enables highly multiplexed imaging [scRNA-seq]

2024-04-21 | GSE242814 | GEO

Pelagic diatoms communicate through synchronized beacon natural fluorescence signaling.

Project description:[Figure: see text].

| S-EPMC8673755 | biostudies-literature

Fluorescence-detected linear dichroism imaging in a re-scan confocal microscope equipped with differential polarization attachment.

Project description:Confocal laser scanning microscopy is probably the most widely used and one of the most powerful techniques in basic biology, medicine and material sciences that is employed to elucidate the architecture of complex cellular structures and molecular macro-assemblies. It has recently been shown that the information content, signal-to-noise ratio and resolution of such microscopes (LSMs) can be improved significantly by adding different attachments or modifying their design, while retaining their user-friendly features and relatively moderate costs. Differential polarization (DP) attachments, using high-frequency modulation/demodulation circuits, have made LSMs capable of high-precision 2D and 3D mapping of the anisotropy of microscopic samples-without interfering with their 'conventional' fluorescence or transmission imaging (Steinbach et al. in Methods Appl Fluoresc 2:015005, 2014). The resolution and the quality of fluorescence imaging have been enhanced in the recently constructed Re-scan confocal microscopy (RCM) (De Luca et al. in Biomed Opt Express 4:2644-2656, 2013). In this work, we developed the RCM technique further, by adding a DP-attachment modulating the exciting laser beam via a liquid crystal (LC) retarder synchronized with the data acquisition system; by this means, and with the aid of a software, fluorescence-detected linear dichroism (FDLD), characteristic of the anisotropic molecular organization of the sample, could be recorded in parallel with the confocal fluorescence imaging. For demonstration, we show FDLD images of a plant cell wall (Ginkgo biloba) stained with Etzold's staining solution.

| S-EPMC6647120 | biostudies-literature

Mechanical distortion of single actin filaments induced by external force: detection by fluorescence imaging.

Project description:Actin is a major component of the cytoskeleton that transmits mechanical stress in both muscle and nonmuscle cells. As the first step toward developing a "bio-nano strain gauge" that would be able to report the mechanical stress imposed on an actin filament, we quantitatively examined the fluorescence intensity of dyes attached to single actin filaments under various tensile forces (5-20 pN). Tensile force was applied via two optically trapped plastic beads covalently coated with chemically modified heavy meromyosin molecules that were attached to both end regions of an actin filament. As a result, we found that the fluorescence intensity of an actin filament, where 20% of monomers were labeled with tetramethylrhodamine (TMR)-5-maleimide at Cys(374) and the filamentous structure was stabilized with nonfluorescent phalloidin, decreased by approximately 6% per 10 pN of the applied force, whereas the fluorescence intensity of an actin filament labeled with either BODIPY TMR cadaverin-iodoacetamide at Cys(374) or rhodamine-phalloidin showed only an approximately 2% decrease per 10 pN of the applied force. On the other hand, spectroscopic measurements of actin solutions showed that the fluorescence intensity of TMR-actin increased 1.65-fold upon polymerization (G-F transformation), whereas that of BODIPY-actin increased only 1.06-fold. These results indicate that the external force distorts the filament structure, such that the microenvironment around Cys(374) approaches that in G-actin. We thus conclude that the fluorescent dye incorporated into an appropriate site of actin can report the mechanical distortion of the binding site, which is a necessary condition for the bio-nano strain gauge.

| S-EPMC2996168 | biostudies-literature

Note: Fast imaging of DNA in atomic force microscopy enabled by a local raster scan algorithm.

Project description:Approaches to high-speed atomic force microscopy typically involve some combination of novel mechanical design to increase the physical bandwidth and advanced controllers to take maximum advantage of the physical capabilities. For certain classes of samples, however, imaging time can be reduced on standard instruments by reducing the amount of measurement that is performed to image the sample. One such technique is the local raster scan algorithm, developed for imaging of string-like samples. Here we provide experimental results on the use of this technique to image DNA samples, demonstrating the efficacy of the scheme and illustrating the order-of-magnitude improvement in imaging time that it provides.

| S-EPMC4048448 | biostudies-other

Robust, coherent, and synchronized circadian clock-controlled oscillations along Anabaena filaments.

Project description:Circadian clocks display remarkable reliability despite significant stochasticity in biomolecular reactions. We study the dynamics of a circadian clock-controlled gene at the individual cell level in Anabaena sp. PCC 7120, a multicellular filamentous cyanobacterium. We found significant synchronization and spatial coherence along filaments, clock coupling due to cell-cell communication, and gating of the cell cycle. Furthermore, we observed low-amplitude circadian oscillatory transcription of kai genes encoding the post-transcriptional core oscillatory circuit and high-amplitude oscillations of rpaA coding for the master regulator transducing the core clock output. Transcriptional oscillations of rpaA suggest an additional level of regulation. A stochastic one-dimensional toy model of coupled clock cores and their phosphorylation states shows that demographic noise can seed stochastic oscillations outside the region where deterministic limit cycles with circadian periods occur. The model reproduces the observed spatio-temporal coherence along filaments and provides a robust description of coupled circadian clocks in a multicellular organism.

| S-EPMC8064755 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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