47 research outputs found
One-shot measurement of the three-dimensional electromagnetic field scattered by a subwavelength aperture tip coupled to the environment
International audienceNear-field scanning optical microscopy (NSOM) achieves subwavelength resolution by bringing a nanosized probe close to the surface of the sample. This extends the spectrum of spatial frequencies that can be detected with respect to a diffraction limited microscope. The interaction of the probe with the sample is expected to affect its radiation to the far field in a way that is often hard to predict. Here we address this question by proposing a general method based on full-field off-axis digital holography microscopy which enables to study in detail the far-field radiation from a NSOM probe as a function of its environment. A first application is demonstrated by performing a three-dimensional (3D) tomographic reconstruction of light scattered from the sub-wavelength aperture tip of a NSOM, in free space or coupled to transparent and plasmonic media. A single holographic image recorded in one shot in the far field contains information on both the amplitude and phase of the scattered light. This is sufficient to reverse numerically the propagation of the electromagnetic field all the way to the aperture tip. Finite Difference Time Domain (FDTD) simulations are performed to compare the experimental results with a superposition of magnetic and electric dipole radiation
From Single-Molecule Interactions to Population-Level Dynamics: Understanding the Complex Organization of RNA Pol II in the Nucleus of Living Cells
Transcription involves a complex exchange within a reservoir of proteins in the nucleoplasm, and the specific recruitment of individual proteins at specific gene loci. However, understanding the spatial distribution of individual proteins and the temporal behavior in the nucleus of living cells remains challenging. Using 3D super-resolution fluorescence microscopy and cluster analysis, we observe that the distribution of RNA Polymerase II (Pol II) cluster sizes, measured as the number of polymerases per cluster, follows a â3/2 power law. Radial dependent analysis of the spatial distribution of Pol II also shows scale-invariance, consistent with a so-called self-organized criticality in a fractal geometry of dimension âŒ2.7. These results suggest a diffusion-based mechanism whereby, via transient interactions, massive recruitment and dismissal of pol II molecules can occur at specific loci in the nucleoplasm. Kinetic measurements using single-molecule detection in live cells reveal Pol II binding dynamics within minutes. Serum-induced transcription increased Pol II binding kinetics in live cells by an order of magnitude. Together, these results provide a comprehensive view of the spatio-temporal organization of Pol II in the nucleus: from the global population distribution, to single molecule recruitment at specific loci in live cells. This comprehensive single-cell approach can be adopted for other proteins beside RNA Pol II, for real-time quantification of protein organization in vivo, with single-molecule sensitivity
Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells
Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cellâs ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation
Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus
Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.Nikon France (Research contract)France. Agence nationale de la recherche (PCV DYNAFT 08-PCVI-0013)France. Agence nationale de la recherche (DynamIC ANR-12-BSV5-0018)Fondation pour la recherche meÌdicaleNetherlands Organization for Scientific Research (Rubicon fellowship
Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus
Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time