We demonstrate circular dichroism (CD) in the second harmonic generation (SHG) signal from chiral assemblies of G-shaped nanostructures made of gold. The arrangement of the G shapes is crucial since upon reordering them the SHG-CD effect disappears. Microscopy reveals SHG "hotspots" assemblies, which originate in enantiomerically sensitive plasmon modes, having the novel property of exhibiting a chiral geometry themselves in relation with the handedness of the material. These results open new frontiers in studying chirality.

Fluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure-interaction-diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.

Inter-laboratory comparison of nanoparticle size measurements using dynamic light scattering and differential centrifugal sedimentation. NanoImpact, (2018).

Here we describe a new BODIPY-based membrane probe (1) that provides an alternative to dialkylcarbocyanine dyes, such as DiI-C18, that can be excited in the blue spectral region. Compound 1 has unbranched octadecyl chains at the 3,5-positions and a meso-amino function. In organic solvents, the absorption and emission maxima of 1 are determined mainly by solvent acidity and dipolarity. The fluorescence quantum yield is high and reaches 0.93 in 2-propanol. The fluorescence decays are well fitted with a single-exponential in pure solvents and in small and giant unilamellar vesicles (GUV) with a lifetime of ca. 4 ns. Probe 1 partitions in the same lipid phase as DiI-C18(5) for lipid mixtures containing sphingomyelin and for binary mixtures of dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC). The lipid phase has no effect on the fluorescence lifetime but influences the fluorescence anisotropy. The translational diffusion coefficients of 1 in GUVs and OLN-93 cells are of the same order as those reported for DiI-C18. The directions of the absorption and emission transition dipole moments of 1 are calculated to be parallel. This is reflected in the high steady-state fluorescence anisotropy of 1 in high ordered lipid phases. Molecular dynamic simulations of 1 in a model of the DOPC bilayer indicate that the average angle of the transition moments with respect to membrane normal is ca. 70°, which is comparable with the value reported for DiI-C18.

The heterogeneity in composition and interaction within the cellular membrane translates into a wide range of diffusion coefficients of its constituents. Therefore, several complementary microfluorimetric techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and singleparticle tracking (SPT) have to be applied to explore the dynamics of membrane components. The recently introduced raster image correlation spectroscopy (RICS) offers a much wider dynamic range than each of these methods separately and allows for spatial mapping of the dynamic properties. RICS is implemented on a confocal laser-scanning microscope (CLSM), and the wide dynamic range is achieved by exploiting the inherent time information carried by the scanning laser beam in the generation of the confocal images. The original introduction of RICS used two-photon excitation and photon counting detection. However, most CLSM systems are based on one-photon excitation with analog detection. Here we report on the performance of such a commercial CLSM (Zeiss LSM 510 META) in the study of the diffusion of the fluorescent lipid analog 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethyl-indodicarbocyanine perchlorate (DiI-C 18 (5)) both in giant unilamellar vesicles and in the plasma membrane of living oligodendrocytes, i.e., the myelin-producing cells of the central nervous system. It is shown that RICS on a commercial CLSM with analog detection allows for reliable results in the study of membrane diffusion by removal of unwanted correlations introduced by the analog detection system. The results obtained compare well with those collected by FRAP and FCS.