Super-resolution fluorescence microscopy can be achieved by image reconstruction after spatially patterned illumination or sequential photo-switching and read-out. Reconstruction algorithms and microscope performance are typically tested using simulated image data, due to a lack of strategies to pattern complex fluorescent patterns with nanoscale dimension control. Here, we report direct electron-beam patterning of fluorescence nanopatterns as calibration standards for super-resolution fluorescence. Patterned regions are identified with both electron microscopy and fluorescence labelling of choice, allowing precise correlation of predefined pattern dimensions, a posteriori obtained electron images, and reconstructed super-resolution images.

Focal adhesions (FAs) are the main cellular structures to link the intracellular cytoskeleton to the extracellular matrix. FAs mediate cell adhesion, are important for cell migration and are involved in many (patho)-physiological processes. Here we examined FAs and their associated actin fibres using correlative fluorescence and scanning electron microscopy (SEM). We used fluorescence images of cells expressing paxillin-GFP to define the boundaries of FA complexes in SEM images, without using SEM contrast enhancing stains. We observed that SEM contrast was increased around the actin fibre entry site in 98% of FAs, indicating increases in protein density and possibly also phosphorylation levels in this area. In nearly three quarters of the FAs, these nanostructures had a fork shape, with the actin forming the stem and the high-contrast FA areas the fork. In conclusion, the combination of fluorescent and electron microscopy allowed accurate localisation of a highly abundant, novel fork structure at the FA-actin interface.

Super-resolution structured illumination microscopy (SIM) has become a widely used method for biological imaging. Standard reconstruction algorithms, however, are prone to generate noise-specific artifacts that limit their applicability for lower signal-to-noise data. Here we present a physically realistic noise model that explains the structured noise artifact, which we then use to motivate new complementary reconstruction approaches. True-Wiener-filtered SIM optimizes contrast given the available signal-to-noise ratio, and flat-noise SIM fully overcomes the structured noise artifact while maintaining resolving power. Both methods eliminate ad hoc user-adjustable reconstruction parameters in favor of physical parameters, enhancing objectivity. The new reconstructions point to a trade-off between contrast and a natural noise appearance. This trade-off can be partly overcome by further notch filtering but at the expense of a decrease in signal-to-noise ratio. The benefits of the proposed approaches are demonstrated on focal adhesion and tubulin samples in two and three dimensions, and on nanofabricated fluorescent test patterns.

Polymersomes have the potential to be applied in targeted alpha radionuclide therapy, while in addition preventing release of recoiling daughter isotopes. In this study, we investigated the cellular uptake, post uptake processing and intracellular localization of polymersomes. Methods: High-content microscopy was used to validate polymersome uptake kinetics. Confocal (live cell) microscopy was used to elucidate the uptake mechanism and DNA damage induction. Intracellular distribution of polymersomes in 3-D was determined using super-resolution microscopy. Results: We found that altering polymersome size and concentration affects the initial uptake and overall uptake capacity; uptake efficiency and eventual plateau levels varied between cell lines; and mitotic cells show increased uptake. Intracellular polymersomes were transported along microtubules in a fast and dynamic manner. Endocytic uptake of polymersomes was evidenced through co-localization with endocytic pathway components. Finally, we show the intracellular distribution of polymersomes in 3-D and DNA damage inducing capabilities of²¹³Bi labeled polymersomes. Conclusion: Polymersome size and concentration affect the uptake efficiency, which also varies for different cell types. In addition, we present advanced assays to investigate uptake characteristics in detail, a necessity for optimization of nano-carriers. Moreover, by elucidating the uptake mechanism, as well as uptake extent and geometrical distribution of radiolabeled polymersomes we provide insight on how to improve polymersome design.

Structured illumination microscopy (SIM) for the imaging of alpha particle tracks in fluorescent nuclear track detectors (FNTD) was evaluated and compared to confocal laser scanning microscopy (CLSM). FNTDs were irradiated with an external alpha source and imaged using both methodologies. SIM imaging resulted in improved resolution, without increase in scan time. Alpha particle energy estimation based on the track length, direction and intensity produced results in good agreement with the expected alpha particle energy distribution. A pronounced difference was seen in the spatial scattering of alpha particles in the detectors, where SIM showed an almost 50% reduction compared to CLSM. The improved resolution of SIM allows for more detailed studies of the tracks induced by ionising particles. The combination of SIM and FNTDs for alpha radiation paves the way for affordable and fast alpha spectroscopy and dosimetry. Journal compilation