Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells.

RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70–90% for in vivo imaging. Also, we extended the information content of the images by acquiring the complete temporal evolution of the fluorescence generated by reversible switchable fluorescent proteins. This generates a series of images with different spatial resolution and SNR, from conventional to RESOLFT images, which combined through a multi-image deconvolution algorithm further enhances the effective resolution. We reported nanoscale imaging of organelles up to 35 Hz and actin dynamics during an invasion process at a depth of 20–30 µm inside a living Caenorhabditis elegans worm.

Super-resolution fluorescence microscopy allows for unprecedented in situ visualization of biological structures, but its application to materials science has so far been comparatively limited. One of the main reasons is the lack of powerful dyes that allow for labeling and photoswitching in materials science systems. In this study it is shown that appropriate substitution of diarylethenes bearing a fluorescent closed and dark open form paves the way for imaging nanostructured materials with three of the most popular super-resolution fluorescence microscopy methods that are based on different concepts to achieve imaging beyond the diffraction limit of light. The key to obtain optimal resolution lies in a proper control over the photochemistry of the photoswitches and its adaption to the system to be imaged. It is hoped that the present work will provide researchers with a guide to choose the best photoswitch derivative for super-resolution microscopy in materials science, just like the correct choice of a Swiss Army Knife's tool is essential to fulfill a given task.

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Complex motor skills take considerable time and practice to learn. Without continued practice the level of skill performance quickly degrades, posing a problem for the timely utilization of skilled motor behaviors. Here we quantified the recurring development of vocal motor skills and the accompanying changes in synaptic connectivity in the brain of a songbird, while manipulating skill performance by consecutively administrating and withdrawing testosterone. We demonstrate that a songbird with prior singing experience can significantly accelerate the re-acquisition of vocal performance. We further demonstrate that an increase in vocal performance is accompanied by a pronounced synaptic pruning in the forebrain vocal motor area HVC, a reduction that is not reversed when birds stop singing. These results provide evidence that lasting synaptic changes in the motor circuitry are associated with the savings of motor skills, enabling a rapid recovery of motor performance under environmental time constraints.

In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers.

SignificanceBasement membrane is a particular kind of sheet-like extracellular matrix that separates tissue compartments. Invasion of cells through basement membrane barriers is a key aspect of many normal and pathological processes, including organ development and cancer cell metastasis. Invasive protrusions are rich in actin, a cytoskeletal biopolymer, the self-assembly of which can produce force and remodel extracellular matrix. However, in the invasive protrusion, actin has been attributed a function in scaffolding and trafficking, and its mechanical role has not been explored. Here we show that invading cells in C. elegans apply forces via actin assembly to break through basement membrane, and that force production is one of the key features of invasion.

The tilted gel phase of lipid bilayers can display in-plane orientational texture due to long-range alignment of the molecular director. We explore systematic variations of texture defects in a series of binary phospholipid membranes. Using polarized two-photon fluorescence microscopy, the texture pattern of single domains is revealed. The appearance of a central vortex-type defect in each domain correlates with a particular range of hydrophobic mismatch values h > 1 nm at the domain border while domains with h < 1 nm correlate with uniformly aligned texture. The central vortex defect is characterized by a defect angle, indicating its bend or splay nature. Using image analysis, we measure the defect angle and find that it has primarily bend character for small mismatch values (h ≈ 1 nm) and primarily splay nature for larger values of h. For domains containing a vortex, the domain shape is decoupled from the texture while for uniformly textured domains there is a preferred texture orientation of ≃45° along the domain border. The results establish a foundation for understanding texture phenomena in compositionally complex membranes.