This tutorial review summarizes the most important results and developments in the field of polymer science by means of single molecule fluorescence spectroscopy (SMFS) at ambient temperatures. A broad range of topics will be addressed and it will be discussed which single molecule methods are suitable to get the maximum amount of information about polymer structure, polymer dynamics and the photophysics of incorporated or embedded dye molecules. In particular, we will report on the use of polymer films for immobilization of molecules, the visualization of dynamics near the glass transition temperature Tg, the reptation of polymer chains, the conformation adopted by polymer chains and the in situ observation of the polymerization reaction itself.

Single-molecule fluorescence microscopy was used to investigate the dynamics of perylene diimide (PDI) molecules in thin supported polystyrene (PS) films at temperatures up to 135 °C. Such high temperatures, so far unreached in single-molecule spectroscopy studies, were achieved using a custom-built setup which allows for restricting the heated mass to a minimum. This enables temperature-dependent single-molecule fluorescence studies of structural dynamics in the temperature range most relevant to the processing and to applications of thermoplastic materials. In order to ensure that polymer chains were relaxed, a molecular weight of 3000 g/mol, clearly below the entanglement length of PS, was chosen. We found significant heterogeneities in the motion of single PDI probe molecules near T(g). An analysis of the track radius of the recorded single-probe molecule tracks allowed for a distinction between mobile and immobile molecules. Up to the glass transition temperature in bulk, T(g,bulk), probe molecules were immobile; at temperatures higher than T(g,bulk) + 40 K, all probe molecules were mobile. In the range between 0 and 40 K above T(g,bulk) the fraction of mobile probe molecules strongly depends on film thickness. In 30-nm thin films mobility is observed at lower temperatures than in thick films. The fractions of mobile probe molecules were compared and rationalized using Monte Carlo random walk simulations. Results of these simulations indicate that the observed heterogeneities can be explained by a model which assumes a T(g) profile and an increased probability of probe molecules remaining at the surface, both effects caused by a density profile with decreasing polymer density at the polymer-air interface.

Compartmentalization in soft matter is important for segregating and coordinating chemical reactions, sequestering (re)active components, and integrating multifunctionality. Advances depend crucially on quantitative 3D visualization in situ with high spatiotemporal resolution. Here, we show the direct visualization of different compartments within adaptive microgels using a combination of in situ electron and super-resolved fluorescence microscopy. We unravel new levels of structural details and address the challenge of reconstructing 3D information from 2D projections for nonuniform soft matter as opposed to monodisperse proteins. Moreover, we visualize the thermally induced shrinkage of responsive core-shell microgels live in water. This strategy opens doors for systematic in situ studies of soft matter systems and their application as smart materials.

The in situ imaging of soft matter is of paramount importance for a detailed understanding of functionality on the nanoscopic scale. Although super-resolution fluorescence microscopy methods with their unprecedented imaging capabilities have revolutionized research in the life sciences, this potential has been far less exploited in materials science. One of the main obstacles for a more universal application of superresolved fluorescence microscopy methods is the limitation of readily available suitable dyes to overcome the diffraction limit. Here, we report a novel diarylethene-based photoswitch with a highly fluorescent closed and a nonfluorescent open form. Its photophysical properties, switching behavior, and high photostability make the dye an ideal candidate for photoactivation localization microscopy (PALM). It is capable of resolving apolar structures with an accuracy far beyond the diffraction limit of optical light in cylindrical micelles formed by amphiphilic block copolymers.The nanoscopic structure of soft-matter materials determines their properties. [1] Therefore, methods to directly visualize structures in the nanometer range are of paramount importance for the ongoing evolution of novel materials with specialized and adaptive properties for sophisticated applications. Scanning probe microscopy techniques give access to the nanometer range and determine surface properties such as topology and softness, [2,3] while modern electron microscopy methods, such as scanning electron microscopy (SEM) [4,5] and transmission electron microscopy (TEM), [6][7][8] can yield structural information even in the subnanometer range when there is sufficient electron density contrast. Despite the success of these methods, they are technically demanding and time-consuming. Furthermore, many softmatter samples possess poor electron contrast, and require non-invasive in situ imaging below the surface as well as the possibility to directly study dynamics. In recent years, superresolved fluorescence microscopy has revolutionized optical imaging, [9][10][11][12][13][14] by utilizing the photophysical or photochemical switching of fluorescent dyes in a sophisticated manner in combination with modern optics. So far, the life sciences have benefited, in particular, from the new possibilities of resolving structures well beyond the diffraction limit of light. Only a few examples of the application of super-resolution microscopy to materials science have been reported, [15][16][17][18] since concepts that require, for example, the addition of (polar) switching buffers often fail for these systems. Therefore, the main bottleneck for more universal applications of super-resolution imaging are switchable dyes with suitable (photo-)physical and chemical properties, such as high photostability, adjustable switching rates, minimum interaction with the environment to be probed, and simple design, with the possibility of multiple and straightforward derivatization for the specific labeling of structures or compartments. [19] ...

Electroporation is a physical method of transferring molecules into cells and tissues. It takes advantage of the transient permeabilization of the cell membrane induced by electric field pulses, which gives hydrophilic molecules access to the cytoplasm. This method offers high transfer efficiency for small molecules that freely diffuse through electrically permeabilized membranes. Larger molecules, such as plasmid DNA, face several barriers (plasma membrane, cytoplasmic crowding, and nuclear envelope), which reduce transfection efficiency and engender a complex mechanism of transfer. Our work provides insight into the way electrotransferred DNA crosses the cytoplasm to reach the nucleus. For this purpose, single-particle tracking experiments of fluorescently labeled DNA were performed. Investigations were focused on the involvement of the cytoskeleton using drugs disrupting or stabilizing actin and tubulin filaments as the two relevant cellular networks for particle transport. The analysis of 315 movies (~4,000 trajectories) reveals that DNA is actively transported through the cytoskeleton. The large number of events allows a statistical quantification of the DNA motion kinetics inside the cell. Disruption of both filament types reduces occurrence and velocities of active transport and displacements of DNA particles. Interestingly, stabilization of both networks does not enhance DNA transport.