Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Intramolecular fluorescence correlation spectroscopy in a feedback tracking microscope.

ABSTRACT: We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that in comparison to traditional fluorescence correlation spectroscopy, tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity, and long-timescale decays. We demonstrate our approach using a three-dimensional tracking microscope to study genomic lambda-phage DNA molecules with various fluorescence label configurations.

SUBMITTER: McHale K

PROVIDER: S-EPMC2895373 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Intramolecular fluorescence correlation spectroscopy in a feedback tracking microscope.

McHale Kevin K Mabuchi Hideo H

Biophysical journal 20100701 1

We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that in comparison to traditional fluorescence correlation spectroscopy, tracking provides enhanced sensitivity to translational diffusion, mo ...[more]

PMID: 20655860

Similar Datasets

Detecting intramolecular dynamics and multiple Forster resonance energy transfer states by fluorescence correlation spectroscopy.

Project description:Fluorescence correlation spectroscopy (FCS) is a robust method for the detection of intramolecular dynamics in proteins but is also susceptible to interference from other dynamic processes such as triplet kinetics and photobleaching. We describe an approach for the detection of intramolecular dynamics in proteins labeled with a FRET dye pair based on global fitting to the two autocorrelation functions (green-green and red-red) and the two cross-correlation functions (green-red and red-green). We applied the method to detect intramolecular dynamics in the Ca(2+) signaling protein calmodulin. Dynamics were detected on the 100 mus time scale in Ca(2+)-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro(15)-Cys) demonstrate the reliability of the method for isolating intramolecular dynamics from other dynamic processes on the microsecond time scale and confirm the absence of intramolecular dynamics of polyproline. We further show the sensitivity of the initial amplitudes of the FCS auto- and cross-correlation functions to the presence of multiple FRET states, static or dynamic. The FCS measurements also show that the diffusion of Ca(2+)-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion.

| S-EPMC2910717 | biostudies-literature

Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy.

Project description:To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hr. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development.

| S-EPMC4946880 | biostudies-literature

Interferometric fluorescence cross correlation spectroscopy.

Project description:Measuring transport properties like diffusion and directional flow is essential for understanding dynamics within heterogeneous systems including living cells and novel materials. Fluorescent molecules traveling within these inhomogeneous environments under the forces of Brownian motion and flow exhibit fluctuations in their concentration, which are directly linked to the transport properties. We present a method utilizing single photon interference and fluorescence correlation spectroscopy (FCS) to simultaneously measure transport of fluorescent molecules within aqueous samples. Our method, within seconds, measures transport in thousands of homogenous voxels (100 nm)3 and under certain conditions, eliminates photo-physical artifacts associated with blinking of fluorescent molecules. A comprehensive theoretical framework is presented and validated by measuring transport of quantum dots, associated with VSV-G receptor along cellular membranes as well as within viscous gels.

| S-EPMC6919592 | biostudies-literature

An alternative framework for fluorescence correlation spectroscopy.

Project description:Fluorescence correlation spectroscopy (FCS), is a widely used tool routinely exploited for in vivo and in vitro applications. While FCS provides estimates of dynamical quantities, such as diffusion coefficients, it demands high signal to noise ratios and long time traces, typically in the minute range. In principle, the same information can be extracted from microseconds to seconds long time traces; however, an appropriate analysis method is missing. To overcome these limitations, we adapt novel tools inspired by Bayesian non-parametrics, which starts from the direct analysis of the observed photon counts. With this approach, we are able to analyze time traces, which are too short to be analyzed by existing methods, including FCS. Our new analysis extends the capability of single molecule fluorescence confocal microscopy approaches to probe processes several orders of magnitude faster and permits a reduction of photo-toxic effects on living samples induced by long periods of light exposure.

| S-EPMC6694112 | biostudies-literature

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

Project description:Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

| S-EPMC2582625 | biostudies-literature

The spectroscopic basis of fluorescence triple correlation spectroscopy.

Project description:We have developed fluorescence triple correlation spectroscopy (F3CS) as an extension of the widely used fluorescence microscopy technique fluorescence correlation spectroscopy. F3CS correlates three signals at once and provides additional capabilities for the study of systems with complex stoichiometry, kinetic processes, and irreversible reactions. A general theory of F3CS was developed to describe the interplay of molecular dynamics and microscope optics, leading to an analytical function to predict experimental triple correlations of molecules that freely diffuse through the tight focus of the microscope. Experimental correlations were calculated from raw fluorescence data using triple correlation integrals that extend multiple-tau correlation theory to delay times in two dimensions. The quality of experimental data was improved by tuning specific spectroscopic parameters and employing multiple independent detectors to minimize optoelectronic artifacts. Experiments with the reversible system of freely diffusing 16S rRNA revealed that triple correlation functions contain symmetries predicted from time-reversal arguments. Irreversible systems are shown to break these symmetries, and correlation strategies were developed to detect time-reversal asymmetries in a comprehensive way with respect to two delay times, each spanning many orders of magnitude in time. The correlation strategies, experimental approaches, and theory developed here enable studies of the composition and dynamics of complex systems using F3CS.

| S-EPMC3293655 | biostudies-literature

MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope.

Project description:The recently introduced minimal photon fluxes (MINFLUX) concept pushed the resolution of fluorescence microscopy to molecular dimensions. Initial demonstrations relied on custom made, specialized microscopes, raising the question of the method's general availability. Here, we show that MINFLUX implemented with a standard microscope stand can attain 1-3 nm resolution in three dimensions, rendering fluorescence microscopy with molecule-scale resolution widely applicable. Advances, such as synchronized electro-optical and galvanometric beam steering and a stabilization that locks the sample position to sub-nanometer precision with respect to the stand, ensure nanometer-precise and accurate real-time localization of individually activated fluorophores. In our MINFLUX imaging of cell- and neurobiological samples, ~800 detected photons suffice to attain a localization precision of 2.2 nm, whereas ~2500 photons yield precisions <1 nm (standard deviation). We further demonstrate 3D imaging with localization precision of ~2.4 nm in the focal plane and ~1.9 nm along the optic axis. Localizing with a precision of <20 nm within ~100 µs, we establish this spatio-temporal resolution in single fluorophore tracking and apply it to the diffusion of single labeled lipids in lipid-bilayer model membranes.

| S-EPMC7935904 | biostudies-literature

Spectrally resolved fluorescence correlation spectroscopy based on global analysis.

Project description:Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto- and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra.

| S-EPMC5780552 | biostudies-literature

Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Concepts and Applications.

Project description:We review the basic concepts and recent applications of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which is the extension of fluorescence correlation spectroscopy (FCS) to analyze the correlation of fluorescence lifetime in addition to fluorescence intensity. Fluorescence lifetime is sensitive to the microenvironment and can be a "molecular ruler" when combined with FRET. Utilization of fluorescence lifetime in 2D FLCS thus enables us to quantify the inhomogeneity of the system and the interconversion dynamics among different species with a higher time resolution than other single-molecule techniques. Recent applications of 2D FLCS to various biological systems demonstrate that 2D FLCS is a unique and promising tool to quantitatively analyze the microsecond conformational dynamics of macromolecules at the single-molecule level.

| S-EPMC6278346 | biostudies-literature

Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy.

Project description:Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of approximately 100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved.

| S-EPMC2084225 | 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