Single fluorescent molecules, acting as ideal point dipoles, offer a unique means to probe light-matter interactions at the nanoscale. Here, we exploit this property to map the chiral and vectorial structure of tightly focused optical fields using individual, immobilized terrylene diimide molecules. By scanning the excitation focus under linear and circular polarization, we obtain three-dimensional fluorescence excitation maps that directly visualize the handedness and symmetry breaking inherent to circularly polarized light. The measured patterns show excellent quantitative agreement with a full vectorial diffraction model, enabling the accurate determination of both molecular orientations and the local field structure. This approach establishes single molecules as quantitative nanoprobes of optical chirality, offering new strategies for characterizing complex light fields and polarization effects in nanophotonic, plasmonic, and anisotropic materials.

Fluorescence lifetimes near dielectric interfaces depend on molecular dipole orientation, but this relationship becomes more complex when emitters are confined within finite nanoscopic films. Here, we combine radially polarized excitation imaging with time-correlated single-photon counting to follow the orientation and fluorescence lifetime of individual PDI molecules undergoing rotational motion in thin poly(n-butyl methacrylate) films. Orientation-sensitive fluorescence patterns were analyzed using vectorial wave-optical simulations, enabling dipole orientation and excited-state lifetime to be correlated over time for the same emitter. The lifetime–orientation relationships vary systematically with film thickness and between molecules within the same film. Electrodynamic calculations for the glass–polymer–air geometry reproduce the principal trends and show that the observed heterogeneity is consistent with different axial emitter positions. These results establish single molecular dipoles as probes of finite nanoscopic dielectric environments.

Abstract Transmembrane signaling mediated by cytokine receptors orchestrates key cellular processes such as proliferation, differentiation, and immune responses. While numerous high-resolution structures of cytokine receptor ectodomains are available, the structural organization of the largely disordered intracellular domain (ICD) has remained unclear. Here, we interrogate the axial organization of cytokine receptor signaling complexes at the plasma membrane by metal-induced energy transfer (MIET). For this purpose, we leveraged biofunctionalized nanodot arrays (bNDAs) to capture cell surface receptors at a defined distance from the substrate. Readout by fluorescence lifetime imaging microscopy enabled quantifying axial distances of proteins in the plasma membrane of cells at both ensemble and single-molecule levels with a resolution of ∼1 nm. Using the prototypic, biomedically relevant class I cytokine receptor GP130 as a model system, we uncover by MIET that the ICD extends randomly into the cytosol in the resting state, but surprisingly undergoes an axial compaction upon signal activation. These results demonstrate the potential of bNDA-supported MIET for resolving the axial architecture of signaling complexes within the cellular context.

In the burgeoning field of super-resolution fluorescence microscopy, significant efforts are being dedicated to expanding its applications into the 3D domain. Various methodologies have been developed that enable isotropic resolution at the nanometer scale, facilitating the visualization of 3D subcellular structures with unprecedented clarity. Central to this progress is the need for reliable 3D structures that are biologically compatible for validating resolution capabilities. Choosing the optimal standard poses a considerable challenge, necessitating, among other attributes, precisely defined geometry and the capability for specific labeling at sub-diffraction-limit distances. In this context, the use of the non-human-infecting virus, bacteriophage T4 is introduced as an effective and straightforward bio-ruler for 3D super-resolution imaging. Employing DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) along with the technique of astigmatic imaging, the icosahedral capsid of the bacteriophage T4, measuring 120 nm in length and 86 nm in width, and its hollow viral tail is uncovered. This level of detail in light microscopy represents a significant advancement in T4 imaging. A simple protocol for the production and preparation of samples is further outlined. Moreover, the extensive potential of bacteriophage T4 as a multifaceted 3D bio-ruler, proposing its application as a novel benchmark for 3D super-resolution imaging in biological studies is explored.

We present a comprehensive review of super-resolution optical fluctuation imaging (SOFI), a robust technique that leverages temporal fluctuations in fluorescence intensity to achieve super-resolution imaging without the need for single-molecule localization. The Review starts with a historical overview of super-resolution microscopy techniques, and then focuses on SOFI’s core principle—the analysis of intensity fluctuations using cumulants to improve spatial resolution. The paper discusses technical challenges, such as photobleaching, blinking kinetics and pixel size limitations, as well as proposing solutions like Fourier upsampling and balanced SOFI to mitigate these issues. Additionally, we discuss potential advancements in the field, including the integration of SOFI with other super-resolution modalities like structured illumination microscopy and image scanning microscopy, and the application of SOFI in cryo-fluorescence microscopy and quantum emitter-based imaging. This paper aims to serve as an essential resource for researchers interested in utilizing SOFI for high-resolution imaging in diverse biological applications. The authors present a review of super-resolution optical fluctuation imaging (SOFI), including its core working principle, recent advances and remaining challenges.

Over the last 30 years, fluorescence microscopy, renowned for its sensitivity and specificity, has undergone a revolution in resolving ever-smaller details. This advancement began with stimulated emission depletion (STED) microscopy and progressed with techniques such as photoactivatable localization microscopy and stochastic optical reconstruction microscopy (STORM). Single-molecule localization microscopy (SMLM), which encompasses methods like direct STORM, has significantly enhanced image resolution. Even though its speed is slower than that of STED, SMLM achieves higher resolution by overcoming photobleaching limitations, particularly through DNA point accumulation for imaging in nanoscale topography (DNA-PAINT), which continuously renews fluorescent labels. Additionally, cryo-fluorescence microscopy and advanced techniques like minimal photon fluxes imaging (MINFLUX) have pushed the boundaries toward molecular resolution SMLM. This review discusses the latest developments in SMLM, highlighting methods like resolution enhancement by sequential imaging (RESI) and PAINT-MINFLUX and exploring axial localization techniques such as supercritical angle fluorescence and metal-induced energy transfer. These advancements promise to revolutionize fluorescence microscopy, providing resolution comparable to that of electron microscopy.

Single fluorescent molecules, behaving as ideal electric dipole emitters, are powerful nanoscopic probes of complex optical fields. Here, this property is exploited to precisely map the polarization and vectorial structure of tightly focused laser beams, utilizing both linear and circular polarization states. The resulting three-dimensional fluorescence excitation maps strikingly reveal the intrinsic chiral and non-chiral structure of the light field, in perfect quantitative agreement with a full vectorial wave-optical model. This precise correspondence not only enables the reliable determination of absolute molecular orientations but also allows for the accurate characterization of the field's properties. These results fundamentally advance our understanding of light-matter interaction at the single-molecule level and open new avenues for characterizing complex light fields, with broad applications in super-resolution microscopy and nanophotonics.

In the burgeoning field of super-resolution fluorescence microscopy, significant efforts are being dedicated to expanding its applications into the three-dimensional domain. Various methodologies have been developed that enable isotropic resolution at the nanometer scale, facilitating the visualization of three-dimensional subcellular structures with unprecedented clarity. Central to this progress is the need for reliable 3D structures that are biologically compatible for validating resolution capabilities. Choosing the optimal standard poses a considerable challenge, necessitating, among other attributes, precisely defined geometry and the capability for specific labeling at sub-diffraction-limit distances. In this context, we introduce the use of the non-human-infecting virus, bacteriophage T4, as an effective and straightforward bio-ruler for three-dimensional super-resolution imaging. Employing DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) along with the technique of astigmatic imaging, we uncover the icosahedral capsid of the bacteriophage T4, measuring 120 nm in length and 86 nm in width, and its hollow viral tail. This level of detail in light microscopy represents a significant advancement. We further outline a simple protocol for the production and preparation of samples. Moreover, we explore the extensive potential of bacteriophage T4 as a multi-faceted 3D bio-ruler, proposing its application as a novel benchmark for three-dimensional super-resolution imaging in biological studies. ### Competing Interest Statement The authors have declared no competing interest.

This study introduces a novel solution to the design of structured catalysts, integrating single-piece 3D printing with single-atom catalysis. Structured catalysts are widely employed in industrial processes, as they provide optimal mass and heat transfer, leading to a more efficient use of catalytic materials. They are conventionally prepared using ceramic or metallic bodies, which are then washcoated and impregnated with catalytically active layers. However, this approach may lead to adhesion issues of the latter. By employing photopolymerization printing, a stable and active single-atom catalyst is directly shaped into a stand-alone, single-piece structured material. The battery of characterization methods employed in the present study confirms the uniform distribution of catalytically active species and the structural integrity of the material. Computational fluid dynamics simulations are applied to demonstrate enhanced momentum transfer and light distribution within the structured body. The materials are finally evaluated in the continuous-flow photocatalytic oxidation of benzyl alcohol to benzaldehyde, a relevant reaction to prepare biomass-derived building blocks. The innovative approach reported herein to manufacture a structured single-atom catalyst circumvents the complexities of traditional synthetic methods, offering scalability and efficiency improvements, and highlights the transformative role of 3D printing in catalysis engineering to revolutionize catalysts' design. This work reports a new 3D printing approach for the fabrication of a stable and reusable structured single-atom catalysts via VAT photopolymerization, and the application of the produced material in the continuous-flow photocatalytic oxidation of biomass-derived benzyl alcohol. image

Water molecules play an important role in the structure, function, and dynamics of (bio-) materials. A direct access to the number of water molecules in nanoscopic volumes can thus give new molecular insights into materials and allow for fine-tuning their properties in sophisticated applications. The determination of the local water content has become possible by the finding that H2 O quenches the fluorescence of red-emitting dyes. Since deuterated water, D2 O, does not induce significant fluorescence quenching, fluorescence lifetime measurements performed in different H2 O/D2 O-ratios yield the local water concentration. We combined this effect with the recently developed fluorescence lifetime single molecule localization microscopy imaging (FL-SMLM) in order to nanoscopically determine the local water content in microgels, i.e. soft hydrogel particles consisting of a cross-linked polymer swollen in water. The change in water content of thermo-responsive microgels when changing from their swollen state at room temperature to a collapsed state at elevated temperature could be analyzed. A clear decrease in water content was found that was, to our surprise, rather uniform throughout the entire microgel volume. Only a slightly higher water content around the dye was found in the periphery with respect to the center of the swollen microgels.

In this study, we integrate a single-photon detector array into a confocal laser scanning microscope, enabling the combination of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy. This unique combination delivers a twofold improvement in lateral localization accuracy for single-molecule localization microscopy (SMLM) and maintains its simplicity. Moreover, the addition of lifetime information from our confocal laser scanning microscope eliminates chromatic aberration, particularly crucial for achieving few-nanometre resolution in SMLM. Our approach, named fluorescence-lifetime image scanning microscopy SMLM, is demonstrated through direct stochastic optical reconstruction microscopy and DNA point accumulation for imaging in nanoscale topography experiments on fluorescently labelled cells, showcasing both resolution enhancement and fluorescence-lifetime multiplexing capabilities. The integration of a single-photon detector array and imaging scanning microscopy in a confocal scanning microscope enables doubling the resolution of single-molecule localization microscopy.

The integration of continuous-flow technologies with heterogeneous photocatalysis has recently emerged as a promising strategy for the development of sustainable processes. Although conventional packed bed reactors have been extensively utilized in industrial catalytic applications, they face challenges related to energy transfer in the photocatalytic systems. This study presents an innovative approach to address this issue by integrating heterogeneous photocatalysts with an organic polymer, leading to the fabrication of a mesophotoreactor featuring a bijel-based structural configuration. This novel strategy involves hybridizing polypentadecalactone and in situ confining of carbon nitride in a bicontinuous porous mesoarchitecture. The structural and physicochemical properties of the resulting catalytic composite material are evaluated through an array of characterization methods, affirming the successful integration of carbon nitride within the overall structure. The unique bicontinuous porous architecture of the composite and its suitability for industrial applications is verified, as exemplified by its exceptional efficiency in the photodegradation of methylene blue (>99 %) under flow conditions and remarkable stability up to three reaction cycles. A mathematical model is developed to describe continuous photocatalytic processes occurring in the novel tubular mesophotoreactor, with a specific focus on the degradation of the methylene blue dye. This model is successfully validated, leading to results in agreement with the experimental measurements. Additionally, fluid dynamics simulations demonstrate that the mesophotoreactor design allows for the effective diffusion of light through its channels, resulting in higher irradiation levels compared to conventional systems such as packed bed reactors. The innovative design of the catalytic reactor presented in this work offers a versatile and efficient alternative to the conventional heterogeneous systems, significantly broadening the range of applications for photocatalytic processes.

Super-resolution fluorescence microscopy techniques are powerful tools to investigate polymer systems. In this review, we address how these techniques have been applied to hydrogel nano- and microparticles, so-called nano- or microgels. We outline which research questions on microgels could be addressed and what new insights could be achieved. Studies of the morphology, shape, and deformation of microgels; their internal compartmentalization; the cross-linker distribution and polarity inside them; and their dynamics and diffusion are summarized. In particular, the abilities to super-resolve structures in three dimensions have boosted the research field and have also allowed researchers to obtain impressive 3D images of deformed microgels. Accessing information beyond 3D localization, such as spectral and lifetime properties and correlative imaging or the combination of data with other methods, shines new light onto polymer systems and helps us understand their complexity in detail. Such future trends and developments are also addressed.

Over the past two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution, which is by a factor of 2 to 3 worse. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging, which achieves an axial resolution down to nanometers. It exploits the distance-dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. We combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of subcellular structures. Moreover, we used spectral demixing for implementing dual-color MIET-dSTORM that allows us to image and colocalize, in three dimensions, two different cellular structures simultaneously.

Single-moleculelocalization microscopy (SMLM) at cryogenic temperatureopens new avenues to investigate intact biological samples at thenanoscale and perform cryo-correlative studies. Genetically encodedfluorescent proteins (FPs) are markers of choice for cryo-SMLM, buttheir reduced conformational flexibility below the glass-transitiontemperature hampers efficient cryo-photoswitching. We investigatedcryo-switching of rsEGFP2, one of the most efficient reversibly switchablefluorescent proteins at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visiblemicrospectrophotometry and X-ray crystallography revealed a completelydifferent switching mechanism at & SIM;110 K. At this cryogenictemperature, on-off photoswitching involves the formation oftwo off-states in cis conformation with blue-shiftedabsorption relative to that of the trans protonatedchromophore populated at ambient temperature. Only one of these off-statescan be switched back to the fluorescent on-state by 405 nm light,while both of them are sensitive to UV light at 355 nm. Superior recoveryto the fluorescent on-state by 355 nm light was confirmed at the single-moleculelevel. This suggests, as also shown by simulations, that employing355 nm light in cryo-SMLM experiments using rsEGFP2 and possibly otherFPs could improve the effective labeling efficiency achievable withthis technique. The rsEGFP2 photoswitching mechanism discovered inthis work adds to the panoply of known switching mechanisms in fluorescentproteins.

3D particle tracking and localization provide direct means to monitor biomolecular processes within nano-scale environments. However, optical aberrations due to inhomogeneous refractive indices are a major shortcoming in probing these processes in situ. In particular, point spread functions (PSF) may be distorted resulting in poor localization and linking across frames. This issue is particularly important when using pre-calibrated PSFs that do not take into account sample induced aberrations. The sample induced aberrations are often removed using experimental techniques such as adaptive optics (AO) by introducing new optical components to microscope setups. Here, we propose a computational method, leveraging a Bayesian framework, as an alternative to the AO techniques. Our Bayesian method is capable of simultaneous particle tracking, phase retrieval and PSF reconstruction directly from a given data set, without adding any new hardware to the optical setup. Moreover, our method is data efficient by rigorously propagating uncertainty from all existing sources in the problem, such as the uncertainty in the shape of PSF, often ignored. We benchmark our method using a wide range of synthetic and experimental data.