Exploring Rotational Diffusion with Plasmonic Coupling.

Measuring the orientation dynamics of nanoparticles and nonfluorescent molecules in real time with optical methods is still a challenge in nanoscience and biochemistry. Here, we examine optoplasmonic sensing taking the rotational diffusion of plasmonic nanorods as an experimental model. Our detection method is based on monitoring the dark-field scattering of a relatively large sensor gold nanorod (GNR) (40 nm in diameter and 112 nm in length) as smaller plasmonic nanorods cross its near field. We observe the rotational motion of single small gold nanorods (three samples with about 5 nm in diameter and 15.5, 19.1, and 24.6 nm in length) in real time with a time resolution around 50 ns. Plasmonic coupling enhances the signal of the diffusing gold nanorods, which are 1 order of magnitude smaller in volume (about 300 nm3) than those used in our previous rotational diffusion experiments. We find a better angular sensitivity with plasmonic coupling in comparison to the free diffusion in the confocal volume. Yet, the angle sensitivity we find with plasmonic coupling is reduced compared to the sensitivity expected from simulations at fixed positions due to the simultaneous translational and rotational diffusion of the small nanorods. To get a reliable plasmonic sensor with the full angular sensitivity, it will be necessary to construct a plasmonic assembly with positions and orientations nearly fixed around the optimum geometry.

Loop extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex.

Structural maintenance of chromosomes (SMC) protein complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC family Wadjet complexes structurally resemble the widespread MukBEF genome-organizing complexes but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves circular DNA; however, the molecular mechanism underlying DNA substrate recognition remains unclear. Here, we use in vitro single-molecule imaging to directly visualize DNA loop extrusion and plasmid cleavage by Wadjet. We find that Wadjet is a symmetric DNA loop extruder that simultaneously reels in DNA from both sides of a growing loop and that this activity requires a dimeric JetABC supercomplex containing two dimers of the JetC motor subunit. On surface-anchored plasmid DNAs, Wadjet extrudes the full length of a 44 kilobase pair plasmid, stalls, and then cleaves DNA. Our findings reveal the role of loop extrusion in the specific recognition and elimination of plasmids by Wadjet, and establish loop extrusion as an evolutionarily conserved mechanism among SMC complexes across kingdoms of life.

Burst-by-Burst Measurement of Rotational Diffusion at Nanosecond Resolution Reveals Hot-Brownian Motion and Single-Chain Binding.

We record dark-field scattering bursts of individual gold nanorods, 52 × 15 nm2 in average size, freely diffusing in water suspension. We deduce their Brownian rotational diffusion constant from autocorrelation functions on a single-event basis. Due to spectral selection by the plasmonic resonance with the excitation laser, the distribution of rotational diffusion constants is much narrower than expected from the size distribution measured by TEM. As rotational diffusion depends on particle hydrodynamic volume, viscosity, and temperature, it can sense those parameters at the single-particle level. We demonstrate measurements of hot Brownian rotational diffusion of nanorods in temperature and viscosity gradients caused by plasmonic heating. Further, we monitor hydrodynamic volumes of gold nanorods upon addition of very low concentrations of the water-soluble polymer PVA, which binds to the particles, leading to measurable changes in their diffusion constant corresponding to binding of one to a few polymer coils. We propose this analysis technique for very low concentrations of biomolecules in solution.

The Smc5/6 complex is a DNA loop-extruding motor.

Structural maintenance of chromosomes (SMC) protein complexes are essential for the spatial organization of chromosomes1. Whereas cohesin and condensin organize chromosomes by extrusion of DNA loops, the molecular functions of the third eukaryotic SMC complex, Smc5/6, remain largely unknown2. Using single-molecule imaging, we show that Smc5/6 forms DNA loops by extrusion. Upon ATP hydrolysis, Smc5/6 reels DNA symmetrically into loops at a force-dependent rate of one kilobase pair per second. Smc5/6 extrudes loops in the form of dimers, whereas monomeric Smc5/6 unidirectionally translocates along DNA. We also find that the subunits Nse5 and Nse6 (Nse5/6) act as negative regulators of loop extrusion. Nse5/6 inhibits loop-extrusion initiation by hindering Smc5/6 dimerization but has no influence on ongoing loop extrusion. Our findings reveal functions of Smc5/6 at the molecular level and establish DNA loop extrusion as a conserved mechanism among eukaryotic SMC complexes.

Imaging the Magnetization of Single Magnetite Nanoparticle Clusters via Photothermal Circular Dichroism.

Magnetic imaging is a versatile tool in biological and condensed-matter physics. Existing magnetic imaging techniques either require demanding experimental conditions which restrict the range of their applications or lack the spatial resolution required for single-particle measurements. Here, we combine photothermal (PT) microscopy with magnetic circular dichroism (MCD) to develop a versatile magnetic imaging technique using visible light. Unlike most magnetic imaging techniques, photothermal magnetic circular dichroism (PT MCD) microscopy works particularly well for single nanoparticles immersed in liquids. As a proof of principle, we demonstrate magnetic CD imaging of superparamagnetic magnetite nanoparticulate clusters immersed in microscope immersion oil. The sensitivity of our method allowed us to probe the magnetization curve of single ∼400-nm-diameter magnetite nanoparticulate clusters.

Photothermal Circular Dichroism of Single Nanoparticles Rejecting Linear Dichroism by Dual Modulation.

Circular dichroism (CD) is the property of chiral nanoobjects to absorb circularly polarized light of either handedness to different extents. Photothermal microscopy enables the detection of CD signals with high sensitivity and provides a direct absorptive response of the samples under study. To achieve CD measurements at the single-particle level, one must reduce such artifacts as leakage of linear dichroism (LD) and residual intensity modulation. We have simulated our setup with a simple model, which allows us to tune modulation parameters to obtain a CD signal virtually free from artifacts. We demonstrate the sensitivity of our setup by measuring the very weak inherent CD signals of single gold nanospheres. We furthermore demonstrate that our method can be extended to obtain spectra of the full absorptive properties of single nanoparticles, including isotropic absorption, linear dichroism, and circular dichroism. We then investigate nominally achiral gold nanoparticles immersed in a chiral liquid. Carefully taking into account the intrinsic chirality of the particles and its change due to heat-induced reshaping, we find that the chiral liquid carvone surrounding the particle has no measurable effect on the particles' chirality, down to g-factors of 3 × 10-4.

Photothermal Microscopy: Imaging the Optical Absorption of Single Nanoparticles and Single Molecules.

The photothermal (PT) signal arises from slight changes of the index of refraction in a sample due to absorption of a heating light beam. Refractive index changes are measured with a second probing beam, usually of a different color. In the past two decades, this all-optical detection method has reached the sensitivity of single particles and single molecules, which gave birth to original applications in material science and biology. PT microscopy enables shot-noise-limited detection of individual nanoabsorbers among strong scatterers and circumvents many of the limitations of fluorescence-based detection. This review describes the theoretical basis of PT microscopy, the methodological developments that improved its sensitivity toward single-nanoparticle and single-molecule imaging, and a vast number of applications to single-nanoparticle imaging and tracking in material science and in cellular biology.

Label-Free Plasmonic Detection of Untethered Nanometer-Sized Brownian Particles.

Optical detection of individual nanometer-sized analytes, virus particles, and protein molecules holds great promise for understanding and control of biological samples and healthcare applications. As fluorescent labels impose restrictions on detection bandwidth and require lengthy and invasive processes, label-free optical techniques are highly desirable. Here, we introduce an optical technique capable of transforming gold nanorods commonly used as photostable labels into highly localized high-speed probes. Our method detects single untethered 5 nm diameter gold particles as they traverse subattoliter volumes in Brownian motion with a time resolution below microseconds.