430 research outputs found
A local view on single and coupled molecules
The paper focuses on a novel approach to reveal ultrafast dynamics in single molecules. The main strength of the approach is towards ultrafast processes in extended multi-chromophoric molecular assemblies. Excitonically coupled systems consisting of 2 and 3 rigidly linked perylene-diimide units in a head to tail configuration are studied. Superradiance and inhibited intramolecular decay are observed and discrete jumps in femtosecond response upon break-up of the strong coupling are revealed
Advances in nanophotonics: ultrafast & ultrasensitive
In this tutorial on NanoPhotonics recent advances are highlighted with focus on near field optical methods, ultra-fast probing of single molecules and ultra-sensitive detection of individual non-fluorescent nanoparticles
Near-Field Fluorescence Imaging of Genetic Material: Toward the Molecular Limit
Chromosomes, DNA, and single fluorescent molecules are studied using an aperture-type near-held scanning optical microscope with tuning fork shear force feedback. Fluorescence in situ hybridization labels on repetitive and single copy probes on human metaphase chromosomes are imaged with a width of 80 nm, allowing their localization with nanometer accuracy, in direct correlation with the simultaneously obtained topography. Single fluorophores, both in polymer and covalently attached to amino- silanized glass, are imaged using two-channel fluorescence polarization detection. The molecules are selectively excited according to their dipole orientation. The orientation of the dipole moment of all molecules in one image could be directly determined. Rotational dynamics on a 10-ms to 100-s timescale is observed. Finally, shear force imaging of double-stranded DNA with a vertical sensitivity of 0.2 nm is presented. A DNA height of 1.4 nm is measured, which indicates the nondisturbing character of the shear force mechanism
Weak ergodicity breaking of receptor motion in living cells stemming from random diffusivity
Molecular transport in living systems regulates numerous processes underlying
biological function. Although many cellular components exhibit anomalous
diffusion, only recently has the subdiffusive motion been associated with
nonergodic behavior. These findings have stimulated new questions for their
implications in statistical mechanics and cell biology. Is nonergodicity a
common strategy shared by living systems? Which physical mechanisms generate
it? What are its implications for biological function? Here, we use single
particle tracking to demonstrate that the motion of DC-SIGN, a receptor with
unique pathogen recognition capabilities, reveals nonergodic subdiffusion on
living cell membranes. In contrast to previous studies, this behavior is
incompatible with transient immobilization and therefore it can not be
interpreted according to continuous time random walk theory. We show that the
receptor undergoes changes of diffusivity, consistent with the current view of
the cell membrane as a highly dynamic and diverse environment. Simulations
based on a model of ordinary random walk in complex media quantitatively
reproduce all our observations, pointing toward diffusion heterogeneity as the
cause of DC-SIGN behavior. By studying different receptor mutants, we further
correlate receptor motion to its molecular structure, thus establishing a
strong link between nonergodicity and biological function. These results
underscore the role of disorder in cell membranes and its connection with
function regulation. Due to its generality, our approach offers a framework to
interpret anomalous transport in other complex media where dynamic
heterogeneity might play a major role, such as those found, e.g., in soft
condensed matter, geology and ecology.Comment: 27 pages, 5 figure
An interpretation of fluctuations in enzyme catalysis rate, spectral diffusion, and radiative component of lifetimes in terms of electric field fluctuations
Time-dependent fluctuations in the catalysis rate ({delta}k(t)) observed in single-enzyme experiments were found in a particular study to have an autocorrelation function decaying on the same time scale as that of spectral diffusion {delta}{omega}0(t). To interpret this similarity, the present analysis focuses on a factor in enzyme catalysis, the local electrostatic interaction energy (E) at the active site and its effect on the activation free energy barrier. We consider the slow fluctuations of the electrostatic interaction energy ({delta}E(t)) as a contributor to {delta}k(t) and relate the latter to {delta}{omega}0(t). The resulting relation between {delta}k(t) and {delta}{omega}0(t) is a dynamic analog of the solvatochromism used in interpreting solvent effects on organic reaction rates. The effect of the postulated {delta}E(t) on fluctuations in the radiative component ({delta}{gamma}Formula(t)) of the fluorescence decay of chromophores in proteins also is examined, and a relation between {delta}{gamma}Formula(t) and {delta}{omega}0(t) is obtained. Experimental tests will determine whether the correlation functions for {delta}k(t), {delta}{omega}0(t), and {delta}{gamma}Formula are indeed similar for any enzyme. Measurements of dielectric dispersion, {varepsilon}({omega}), for the enzyme discussed elsewhere will provide further insight into the correlation function for {delta}E(t). They also will determine whether fluctuations in the nonradiative component {gamma}Formula of the lifetime decay has a different origin, fluctuations in distance for example
Quantitative transcription factor binding kinetics at the single-molecule level
We have investigated the binding interaction between the bacteriophage lambda
repressor CI and its target DNA using total internal reflection fluorescence
microscopy. Large, step-wise changes in the intensity of the red fluorescent
protein fused to CI were observed as it associated and dissociated from
individually labeled single molecule DNA targets. The stochastic association
and dissociation were characterized by Poisson statistics. Dark and bright
intervals were measured for thousands of individual events. The exponential
distribution of the intervals allowed direct determination of the association
and dissociation rate constants, ka and kd respectively. We resolved in detail
how ka and kd varied as a function of 3 control parameters, the DNA length L,
the CI dimer concentration, and the binding affinity. Our results show that
although interaction with non-operator DNA sequences are observable, CI binding
to the operator site is not dependent on the length of flanking non-operator
DNA.Comment: 34 pages, 10 figures, accepted by Biophysical Journa
High-density single-molecule maps reveal transient membrane receptor interactions within a dynamically varying environment
Over recent years, super-resolution and single-molecule imaging methods have
delivered unprecedented details on the nanoscale organization and dynamics of
individual molecules in different contexts. Yet, visualizing single-molecule
processes in living cells with the required spatial and temporal resolution
remains highly challenging. Here, we report on an analytical approach that
extracts such information from live-cell single-molecule imaging at
high-labeling densities using standard fluorescence probes. Our
high-density-mapping (HiDenMap) methodology provides single-molecule nanometric
localization accuracy together with millisecond temporal resolution over
extended observation times, delivering multi-scale spatiotemporal data that
report on the interaction of individual molecules with their dynamic
environment. We validated HiDenMaps by simulations of Brownian trajectories in
the presence of patterns that restrict free diffusion with different
probabilities. We further generated and analyzed HiDenMaps from single-molecule
images of transmembrane proteins having different interaction strengths to
cortical actin, including the transmembrane receptor CD44. HiDenMaps uncovered
a highly heterogenous and multi-scale spatiotemporal organization for all the
proteins that interact with the actin cytoskeleton. Notably, CD44 alternated
between periods of random diffusion and transient trapping, likely resulting
from actin-dependent CD44 nanoclustering. Whereas receptor trapping was dynamic
and lasted for hundreds of milliseconds, actin remodeling occurred at the
timescale of tens of seconds, coordinating the assembly and disassembly of CD44
nanoclusters rich regions. Together, our data demonstrate the power of
HiDenMaps to explore how individual molecules interact with and are organized
by their environment in a dynamic fashion.Comment: 33 pages, 5 figure
Broadband plasmonic nanoantennas for multi-color nanoscale dynamics in living cells
Recently, the implementation of plasmonic nanoantennas has opened new
possibilities to investigate the nanoscale dynamics of individual biomolecules
in living cell. However, studies have yet been restricted to single molecular
species as the narrow wavelength resonance of gold-based nanostructures
precludes the simultaneous interrogation of different fluorescently labeled
molecules. Here we exploited broadband aluminum-based nanoantennas carved at
the apex of near-field probes to resolve nanoscale-dynamic molecular
interactions on intact living cell membranes. Through multicolor excitation, we
simultaneously recorded fluorescence fluctuations of dual-color labeled
transmembrane receptors known to form nanoclusters in living cells.
Fluorescence cross-correlation studies revealed transient interactions between
individual receptors in regions of ~60 nm. Moreover, the high
signal-to-background ratio provided by the antenna illumination allowed us to
directly detect fluorescent bursts arising from the passage of individual
receptors underneath the antenna. Remarkably, by reducing the illumination
volume below the characteristic receptor nanocluster sizes, we resolved
molecular diffusion within nanoclusters and distinguished it from nanocluster
diffusion. Spatiotemporal characterization of transient interactions between
molecules is crucial to understand how they communicate with each other to
regulate cell function. Our work demonstrates the potential of broadband
photonic antennas to study multi-molecular events and interactions in living
cell membranes with unprecedented spatiotemporal resolution
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