Meeting experiments at the diffraction barrier: an in-silico widefield fluorescence microscopy

Fluorescence microscopy allows the visualization of live cells and their components, but even with advances in super- resolution microscopy, atomic resolution remains unattainable. On the other hand, molecular simulations (MS) can easily access atomic resolution, but comparison with experimental microscopy images has not been possible. In this work, a novel in-silico widefield fluorescence microscopy is proposed, which reduces the resolution of MS to generate images comparable to experiments. This technique will allow cross-validation and compound the knowledge gained from experiments and MS. We demonstrate that in-silico images can be produced with different optical axis, object focal planes, exposure time, color combinations, resolution, brightness and amount of out-of-focus fluorescence. This allows the generation of images that resemble those obtained from widefield, confocal, light-sheet, two-photon and super-resolution microscopy. This technique not only can be used as a standalone visualization tool for MS, but also lays the foundation for other in-silico microscopy methods.

UCsim2: 2D Structured Illumination Microscopy using UC2

State-of-the-art microscopy techniques enable the imaging of sub-diffraction barrier biological structures at the price of high-costs or lacking transparency. We try to reduce some of these barriers by presenting a super-resolution upgrade to our recently presented open-source optical toolbox UC2. Our new injection moulded parts allow larger builds with higher precision. The 4x lower manufacturing tolerance compared to 3D printing makes assemblies more reproducible. By adding consumer-grade available open-source hardware such as digital mirror devices (DMD) and laser projectors we demonstrate a compact 3D multimodal setup that combines image scanning microscopy (ISM) and structured illumination microscopy (SIM). We demonstrate a gain in resolution and optical sectioning using the two different modes compared to the widefield limit by imaging Alexa Fluor 647- and SiR-stained HeLa cells. We compare different objective lenses and by sharing the designs and manuals of our setup, we make super-resolution imaging available to everyone.

Super-resolution interference lithography enabled by non-equilibrium kinetics of photochromic monolayers

The non-equilibrium kinetics of spirothiopyran monolayers are studied to enable large area interference lithography with feature dimensions that circumvent the diffraction barrier.

Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT

We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5–12-fold compared with their conventional diffraction-limited LS analogs.

Imaging the invisible: resolving cellular microcompartments by superresolution microscopy techniques

Unraveling the spatio-temporal organization of dynamic cellular microcompartments requires live cell imaging techniques capable of resolving submicroscopic structures. While the resolution of traditional far-field fluorescence imaging techniques is limited by the diffraction barrier, several fluorescence-based microscopy techniques providing sub-100 nm resolution have become available during the past decade. Here, we briefly introduce the optical principles of these techniques and compare their capabilities and limitations with respect to spatial and temporal resolution as well as live cell capabilities. Moreover, we summarize how these techniques contributed to a better understanding of plasma membrane microdomains, the dynamic nanoscale organization of neuronal synapses and the sub-compartmentation of microorganisms. Based on these applications, we highlight complementarity of these techniques and their potential to address specific challenges in the context of dynamic cellular microcompartments, as well as the perspectives to overcome current limitations of these methods.