Real-time monitoring of biomechanical activity in aphids by laser speckle contrast imaging.

Studying in vivo feeding and other behaviors of small insects, such as aphids, is important for understanding their lifecycle and interaction with the environment. In this regard, the EPG (electrical penetration graph) technique is widely used to study the feeding activity in aphids. However, it is restricted to recording feeding of single insects and requires wiring insects to an electrode, impeding free movement. Hence, easy and straightforward collective observations, e.g. of groups of aphids on a plant, or probing other aphid activities in various body parts, is not possible. To circumvent these drawbacks, we developed a method based on an optical technique called laser speckle contrast imaging (LSCI). It has the potential for direct, non-invasive and contactless monitoring of a broad range of internal and external activities such as feeding, hemolymph cycling and muscle contractions in aphids or other insects. The method uses a camera and coherent light illumination of the sample. The camera records the laser speckle dynamics due to the scattering and interference of light caused by moving scatters in a probed region of the insect. Analyzing the speckle contrast allowed us to monitor and extract the activity information during aphid feeding on leaves or on artificial medium containing tracer particles. We present evidence that the observed speckle dynamics might be caused by muscle contractions, movement of hemocytes in the circulatory system or food flows in the stylets. This is the first time such a remote sensing method has been applied for optical mapping of the biomechanical activities in aphids.

Single-objective high-resolution confocal light sheet fluorescence microscopy for standard biological sample geometries.

Three-dimensional fluorescence-based imaging of living cells and organisms requires the sample to be exposed to substantial excitation illumination energy, typically causing phototoxicity and photobleaching. Light sheet fluorescence microscopy dramatically reduces phototoxicity, yet most implementations are limited to objective lenses with low numerical aperture and particular sample geometries that are built for specific biological systems. To overcome these limitations, we developed a single-objective light sheet fluorescence system for biological imaging based on axial plane optical microscopy and digital confocal slit detection, using either Bessel or Gaussian beam shapes. Compared to spinning disk confocal microscopy, this system displays similar optical resolution, but a significantly reduced photobleaching at the same signal level. This single-objective light sheet technique is built as an add-on module for standard research microscopes and the technique is compatible with high-numerical aperture oil immersion objectives and standard samples mounted on coverslips. We demonstrate the performance of this technique by imaging three-dimensional dynamic processes, including bacterial biofilm dispersal, the response of biofilms to osmotic shocks, and macrophage phagocytosis of bacterial cells.

Kinetic Control over Self-Assembly of Semiconductor Nanoplatelets.

Semiconductor nanoplatelets exhibit spectrally pure, directional fluorescence. To make polarized light emission accessible and the charge transport effective, nanoplatelets have to be collectively oriented in the solid state. We discovered that the collective nanoplatelets orientation in monolayers can be controlled kinetically by exploiting the solvent evaporation rate in self-assembly at liquid interfaces. Our method avoids insulating additives such as surfactants, making it ideally suited for optoelectronics. The monolayer films with controlled nanoplatelets orientation (edge-up or face-down) exhibit long-range ordering of transition dipole moments and macroscopically polarized light emission. Furthermore, we unveil that the substantial in-plane electronic coupling between nanoplatelets enables charge transport through a single nanoplatelets monolayer, with an efficiency that strongly depends on the orientation of the nanoplatelets. The ability to kinetically control the assembly of nanoplatelets into ordered monolayers with tunable optical and electronic properties paves the way for new applications in optoelectronic devices.

Huge diversity of phleboviruses in ticks from Strandja Nature Park, Bulgaria.

The discovery of the first tick-borne phleboviruses associated with severe disease in humans stimulated studies searching for further previously unknown tick-associated viruses. Novel phleboviruses have subsequently been identified in ticks from the USA, Japan and China and recently also from Europe. Here, we investigated the genetic diversity of tick-borne phleboviruses originating from Strandja Nature Park, Bulgaria, a unique primary forest with evergreen plants that was not affected by the last ice ages in the Pleistocene and Holocene. We found a high genetic diversity of 12 phleboviral sequences in 1542 ticks. The sequences formed five distinct groups and clustered with other tick-borne phleboviruses recently identified in Europe. Although isolation experiments of the detected viruses in cell culture failed, viral RNA copy numbers were stable up to 42 days post infection (dpi) in the supernatant of tick cells whereas they disappeared 14 dpi in that of VeroE6/7 cells. In summary, nearly all tick-associated phleboviruses known to occur in Europe have been detected in one geographic region. Our data show that primary ecosystems in temperate regions are also rich in viral diversity and that this is not only true for tropical regions.

Fluorescence Correlation Spectroscopy in Dilute Polymer Solutions: Effects of Molar Mass Dispersity and the Type of Fluorescent Labeling.

Fluorescence correlation spectroscopy (FCS) has become an important tool in polymer science. Among various other applications the method is often applied to measure the hydrodynamic radius and the degree of fluorescent labeling of polymers in dilute solutions. Here we show that such measurements can be strongly affected by the molar mass dispersity of the studied polymers and the way of labeling. As model systems we used polystyrene and poly(methyl methacrylate) synthesized by atom transfer radical polymerization or free-radical polymerization. Thus, the polymers were either end-labeled bearing one fluorophore per chain or side-labeled with a number of fluorophores per chain proportional to the degree of polymerization.The experimentally measured autocorrelation curves were fitted with a newly derived theoretical model that uses the Schulz-Zimm distribution function to describe the dispersity in the degree of polymerization. For end-labeled polymers having a molecular weight distribution close to Schulz-Zimm, the fits yield values of the number-average degree of polymerization and the polydispersity index similar to those obtained by reference gel permeation chromatography. However, for the side-labeled polymers such fitting becomes unstable, especially for highly polydisperse systems. Brownian dynamic simulations showed that the effect is due to a mutual dependence between the fit parameters, namely, the polydispersity index and the number-average molecular weight. As a consequence, an increase of the polydispersity index can be easily misinterpreted as an increase of the molecular weight when the FCS autocorrelation curves are fitted with a standard single component model, as commonly done in the community.

Photophysical investigation of cyano-substituted terrylenediimide derivatives.

Two new terrylenediimide (TDI) chromophores with cyano substituents in the bay and core area (BCN-TDI and OCN-TDI, respectively) have been characterized by a wide range of techniques, and their applicability for stimulated emission depletion (STED) microscopy has been tested. By cyano substitution an increase of the fluorescence quantum yield and a decrease of the nonradiative rate constant is achieved and attributed to a reduced charge-transfer character of the excited state due to a lower electron density of the TDI core. For BCN-TDI, the substitution in the bay area induces a strong torsional twist in the molecule which, similar to phenoxy bay-perylenediimide (PDI), has a strong effect on the fluorescence lifetime but appears to prevent the aggregation that is observed for OCN-TDI. The single-molecule photobleaching stability of BCN- and OCN-TDI is lower than that of a reference TDI without cyano substitution (C7-TDI), although less so for OCN-TDI. The photophysical properties of the excited singlet state are only slightly influenced by the cyano groups. The observed intense stimulated emission, the pump-dump-probe experiments, and STED single-molecule imaging indicate that STED experiments with the cyano-substituted TDIs are possible. However, because of aggregation and more efficient photobleaching, the performance of BCN- and OCN-TDI is worse than that of the reference compound without cyano groups (C7-TDI). Bay-substituted TDIs are less suitable for STED microscopy.

Hydrodynamic boundary condition of water on hydrophobic surfaces.

By combining total internal reflection fluorescence cross-correlation spectroscopy with Brownian dynamics simulations, we were able to measure the hydrodynamic boundary condition of water flowing over a smooth solid surface with exceptional accuracy. We analyzed the flow of aqueous electrolytes over glass coated with a layer of poly(dimethylsiloxane) (advancing contact angle Θ = 108°) or perfluorosilane (Θ = 113°). Within an error of better than 10 nm the slip length was indistinguishable from zero on all surfaces.

Probing diffusion of single nanoparticles at water-oil interfaces.

The diffusion of nanoparticles at a water-alkane interface is studied using fluorescence correlation spectroscopy. Hydrophilic and hydrophobic quantum dots of 5, 8, and 11 nm radius are used. A slow-down of nanoparticle diffusion at the liquid-liquid interface is observed. The effect is most evident when the viscosities of both liquid phases are similar, here, at the water-decane interface. In this case, the interfacial diffusion coefficients of the hydrophilic particles are 1.5 times and those of the hydrophobic particles 2 times lower than the corresponding bulk values.

Note: An easy way to enable total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) by combining commercial devices for FCS and TIR microscopy.

Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) is a powerful method for studying dynamic processes at liquid-solid interfaces that may have numerous applications in biology, physics, and material science. Despite of its power and versatility, however, the use of TIR-FCS is still rather limited. The main reason for this is the need of a complex, in-house constructed optical setup whose assembly and adjustment is a quite difficult task. Clearly, the availability of ready to use, commercial TIR-FCS setups will strongly boost the application of this important method in many research areas. In this note we show that although such setups are still not available in the market, a proper combination of commercial devices for confocal fluorescence correlation spectroscopy and for total internal reflection microscopy may enable TIR-FCS in a way that do not require any special optical alignments. Furthermore, we demonstrate the capabilities of the setup by measuring the diffusion coefficient of single dye molecule and quantum dots in the very proximity of a water-glass interface.

Direct studies of liquid flows near solid surfaces by total internal reflection fluorescence cross-correlation spectroscopy.

We present a new method to study flow of liquids near solid surface: Total internal reflection fluorescence cross-correlation spectroscopy (TIR-FCCS). Fluorescent tracers flowing with the liquid are excited by evanescent light, produced by epi-illumination through the periphery of a high numerical aperture oil-immersion objective. The time-resolved fluorescence intensity signals from two laterally shifted observation volumes, created by two confocal pinholes are independently measured. The cross-correlation of these signals provides information of the tracers' velocities. By changing the evanescent wave penetration depth, flow profiling at distances less than 200 nm from the interface can be performed. Due to the high sensitivity of the method fluorescent species with different size, down to single dye molecules can be used as tracers. We applied this method to study the flow of aqueous electrolyte solutions near a smooth hydrophilic surface and explored the effect of several important parameters, e.g. tracer size, ionic strength, and distance between the observation volumes.