610 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
Nanoclustering as a dominant feature of plasma membrane organization
Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at nanoscale lengths. In this Commentary, we present selected examples of glycosylphosphatidyl-anchored proteins, Ras family members and several immune receptors that provide evidence for nanoclustering. We advocate the view that nanoclustering is an important part of the hierarchical organization of proteins in the plasma membrane. According to this emerging picture, nanoclusters can be organized on the mesoscale to form microdomains that are capable of supporting cell adhesion, pathogen binding and immune cell-cell recognition amongst other functions. Yet, a number of outstanding issues concerning nanoclusters remain open, including the details of their molecular composition, biogenesis, size, stability, function and regulation. Notions about these details are put forth and suggestions are made about nanocluster function and why this general feature of protein nanoclustering appears to be so prevalent.Postprint (published version
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
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
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