The dynamic behavior of cellular membranes underpins essential biological processes, including signal transduction, intracellular trafficking, and mechanotransduction. However, simultaneously quantifying lateral molecular diffusion and vertical membrane fluctuations in live cells remains challenging. Here, we present dynamic metal-induced energy transfer spectroscopy (dynaMIET), which integrates metal-induced energy transfer with fluorescence correlation spectroscopy to resolve three-dimensional membrane dynamics with nanometer axial sensitivity and microsecond temporal resolution. dynaMIET enables concurrent measurement of lateral diffusion and vertical undulations within a single acquisition. We validate the method using simulations and model membranes and demonstrate its robustness in living cells, applying it to the plasma membrane, endoplasmic reticulum, and nuclear envelope. By capturing both molecular mobility and membrane fluctuations, dynaMIET provides a powerful, noninvasive tool for probing membrane mechanics and organization. This advance opens avenues for studying membrane-associated phenomena in health and disease, including cancer cell mechanics, protein-membrane interactions, and organelle dynamics.

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.

Cell mechanics play a pivotal role in regulating numerous biological processes. Although super-resolution microscopy enables the imaging of cellular forces in the lateral dimension with sub-10-nm resolution, achieving comparable resolution along the axial dimension remains a significant challenge. Here, we introduce metal-induced energy transfer (MIET)-based tension probe microscopy (MIET-TPM), a technique for mapping cellular mechanical forces with nanometer precision in the axial direction. This approach combines the nanometer spatial resolution of MIET imaging with the piconewton sensitivity of DNA-hairpin-based molecular tension probes (MTPs), enabling the simultaneous observation of both the plasma membrane and force-exerting molecules in the axial dimension. Using MIET-TPM, we mapped axial integrin tension within focal adhesions and podosomes, alongside their corresponding plasma membrane height profiles, offering detailed insights into the nanoscale structures and mechanisms involved in force transmission. Notably, MIET-TPM can be implemented on any fluorescence microscopy setup without hardware modifications, making it a versatile and accessible tool that promises to become an integral part of future cellular mechanobiology analysis.

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.

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.

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 dynamics of cellular membranes are crucial for many essential cellular processes. Despite various methods available to study membrane dynamics, simultaneously measuring membrane diffusion and fluctuations within living cell membranes remains challenging. In this study, we developed a method combining metal-induced energy transfer (MIET) with fluorescence correlation spectroscopy (FCS) to accurately measure the three-dimensional (3D) dynamics of membranes, including transverse diffusion and vertical fluctuations. Our approach achieves a spatiotemporal resolution of one nanometer and few microseconds. We demonstrate the efficacy of our technique through experiments on the diffusion and transversal undulations of model membranes. Additionally, we showcase the versatility of MIET-FCS by applying it to various membrane systems within living cells, such as the plasma membrane, endoplasmic reticulum, and nuclear envelope. Our findings provide a powerful tool for investigating complex membrane dynamics in diverse cellular environments, advancing the field of membrane biophysics. ### Competing Interest Statement The authors have declared no competing interest.

A recent addition to the toolbox of super-resolution microscopy methods is fluorescence-lifetime single-molecule localization microscopy (FL-SMLM). The synergy of SMLM and fluorescence-lifetime imaging microscopy (FLIM) combines superior image resolution with lifetime information and can be realized using two complementary experimental approaches: confocal-laser scanning microscopy (CLSM) or wide-field microscopy. Here, we systematically and comprehensively compare these two novel FL-SMLM approaches in different spectral regions. For wide-field FL-SMLM, we use a commercial lifetime camera, and for CLSM-based FL-SMLM we employ a home-built system equipped with a rapid scan unit and a single-photon detector. We characterize the performances of the two systems in localizing single emitters in 3D by combining FL-SMLM with metal-induced energy transfer (MIET) for localization along the third dimension and in the lifetime-based multiplexed bioimaging using DNA-PAINT. Finally, we discuss advantages and disadvantages of wide-field and confocal FL-SMLM and provide practical advice on rational FL-SMLM experiment design.

We report on proof of principle measurements of a concept for a super-resolution imaging method that is based on excitation field density-dependent lifetime modulation of semiconductor nanocrystals. The prerequisite of the technique is access to semiconductor nanocrystals with emission lifetimes that depend on the excitation intensity. Experimentally, the method requires a confocal microscope with fluorescence-lifetime measurement capability that makes it easily accessible to a broad optical imaging community. We demonstrate with single particle imaging that the method allows one to achieve a spatial resolution of the order of several tens of nanometers at moderate fluorescence excitation intensity.

The scientific and educational community is becoming increasingly aware of the impact of current academic working conditions on graduate students’ mental health and how this is affecting scientific progress and ultimately society as a whole. Our study aimed to shed light on the work-related mental health issues affecting graduate students, providing a comprehensive research work including psychological and biological assessment. Our findings showed that a sizeable number of graduate student present anxiety, depression, or high burnout and that the time spent in academia plays an important role. The graduate student population displayed a specific work-related mental health issues profile with an altered hypothalamic–pituitary–adrenal (HPA) axis and low levels of work engagement. Finally, graduate students were equally stressed, with less work engagement, and more anxious and depressed than general workers.

Upon exposure to chronic stressors, how do individuals move from being in a healthy state to a burnout? Strikingly in literature, this has prevailed a categorical view rather than a dimensional one, thus the underlying process that explains the transition from one state to another remains unclear. The aims of the present study are (a) to examine intermediate states between work engagement and burnout using cluster analysis and (b) to examine cortisol differences across these states. Two-hundred and eighty-one Argentine workers completed self-report measures of work engagement and burnout. Salivary cortisol was measured at three time-points: immediately after awakening and 30 and 40min thereafter. Results showed four different states based on the scores in cynicism, exhaustion, vigor, and dedication: engaged, strained, cynical, and burned-out. Cortisol levels were found to be moderate in the engaged state, increased in the strained and cynical states, and decreased in the burned-out state. The increase/decrease in cortisol across the four stages reconciles apparent contradictory findings regarding hypercortisolism and hypocortisolism, and suggests that they may represent different phases in the transition from engagement to burnout. A phase model from engagement to burnout is proposed and future research aimed at evaluating this model is suggested.

The amyloid aggregation of the presynaptic protein α-synuclein (AS) is pathognomonic of Parkinson's disease and other neurodegenerative disorders. Physiologically, AS contributes to synaptic homeostasis by participating in vesicle maintenance, trafficking, and release. Its avidity for highly curved acidic membranes has been related to the distinct chemistry of the N-terminal amphipathic helix adopted upon binding to appropriated lipid interfaces. Pathologically, AS populate a myriad of toxic aggregates ranging from soluble oligomers to insoluble amyloid fibrils. Different gain-of-toxic function mechanisms are linked to prefibrillar oligomers which are considered as the most neurotoxic species. Here, we investigated if amyloid oligomerization could hamper AS function as a membrane curvature sensor. We used fluorescence correlation spectroscopy to quantitatively evaluate the interaction of oligomeric species, produced using a popular method based on lyophilization and rehydration, to lipid vesicles of different curvatures and compositions. We found that AS oligomerization has a profound impact on protein-lipid interaction, altering binding affinity and/or curvature sensitivity depending on membrane composition. Our work provides novel insights into how the formation of prefibrillar intermediate species could contribute to neurodegeneration due to a loss-of-function mechanism. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not dear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS. This article has an associated First Person interview with the first author of the paper.

Autophagy is a constitutive lysosomal catabolic pathway that degrades damaged organelles and protein aggregates. In Parkinson’s disease, the synaptic protein alpha-synuclein (AS) accumulates in neuronal cell bodies and axons. Recent studies indicate that aggregation-prone proteins can spread to other brain cells - such as glia - contributing to progressive deterioration. Although autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, exact mechanisms are not clear and most work was done in neurons and not on microglial cells. Here we report that AS fibrils but not monomers induce lysosomal damage and autophagy in microglial cells and we extensively characterized the dynamics of this response by both live-cell imaging and correlative light-electron microscopy (CLEM). In addition, we found that autophagy inhibition in these cells impairs mitochondrial quality and leads to microglial cell death. We propose that AS accumulation in lysosomes leads to lysosomal damage, which in turn activates canonical autophagy as a rescue mechanism. Our results provide novel findings about the interaction between AS and the autophagy pathway in microglial cells, which may be important for targeting protein misfolding-associated neurodegenerative diseases.

Autophagy is a fundamental cellular homeostatic mechanism, whereby cells autodigest parts of their cytoplasm for removal or turnover. Neurodegenerative disorders are associated with autophagy dysregulation, and drugs modulating autophagy have been successful in several animal models. Microglial cells are phagocytes in the central nervous system (CNS) that become activated in pathological conditions and determine the fate of other neural cells. Here, we studied the effects of autophagy on the production of pro-inflammatory molecules in microglial cells and their effects on neuronal cells. We observed that both trehalose and rapamycin activate autophagy in BV2 microglial cells and down-regulate the production of pro-inflammatory cytokines and nitric oxide (NO), in response to LPS and alpha-synuclein. Autophagy also modulated the phosphorylation of p38 and ERK1/2 MAPKs in BV2 cells, which was required for NO production. These actions of autophagy modified the impact of microglial activation on neuronal cells, leading to suppression of neurotoxicity. Our results demonstrate a novel role for autophagy in the regulation of microglial cell activation and pro-inflammatory molecule secretion, which may be important for the control of inflammatory responses in the CNS and neurotoxicity.

The misfolding and aggregation of the presynaptic protein α-synuclein (AS) into amyloid fibrils is pathognomonic of Parkinson's disease, though the mechanism by which this structural conversion occurs is largely unknown. Soluble oligomeric species that accumulate as intermediates in the process of fibril formation are thought to be highly cytotoxic. Recent studies indicate that oligomer-to-fibril AS transition plays a key role in cell toxicity and progression of neurodegeneration. We previously demonstrated that a subgroup of oligomeric AS species are ordered assemblies possessing a well-defined pattern of intermolecular contacts which are arranged into a distinctive antiparallel β-sheet structure, as opposed to the parallel fibrillar fold. Recently, it was demonstrated that the physiological form of AS is N-terminally acetylated (Ac-AS). Here, we first showed that well-characterized conformational ensembles of Ac-AS, namely monomers, oligomers and fibrils, recapitulate many biophysical features of the nonacetylated protein, such as hydrodynamic, tinctorial, structural and membrane-leakage properties. Then, we relied on ATR-FTIR spectroscopy to explore the structural reorganization during Ac-AS fibrillogenesis. We found that antiparallel β-sheet transient intermediates are built-up at early stages of aggregation, which then evolve to parallel β-sheet fibrils through helix-rich/disordered species. The results are discussed in terms of regions of the protein that might participate in this structural rearrangement. Our work provides new insights into the complex conformational reorganization occurring during Ac-AS amyloid formation.