Spectral information plays a crucial role in biological imaging, yet conventional epifluorescence and histological techniques often rely on RGB image acquisition, limiting the resolution of spectrally overlapping components. Here, we present a phasor-based spectral analysis framework adapted for RGB images, enabling unsupervised segmentation and unmixing without the need for hyperspectral systems or sequential acquisition. By applying a discrete Fourier transform to the red, green, and blue intensities at each pixel, we generate a two-dimensional phasor plot where spectral relationships are encoded in modulation and phase. We demonstrate the utility of this approach across three distinct applications: segmentation of lung histology images stained with hematoxylin and eosin to quantify alveolar collapse, analysis of autofluorescence in skin lesions (nevi and melanoma) to highlight pathological spectral signatures, and spectral unmixing in multicolor-labeled U2OS cells to resolve overlapping fluorophores. Our method improves signal separation, reduces noise, and enhances biological interpretability using standard RGB acquisition. These findings establish RGB phasor analysis as a practical and powerful tool for spectral decomposition and segmentation in microscopy, bridging the gap between conventional imaging and advanced spectral analysis.

Metabolic processes shape ageing and longevity at multiple levels. Emerging evidence shows that many of these processes are orchestrated within and between cellular organelles. Organelles function not only as metabolic reactors but also as signalling hubs, and their coordination plays crucial roles in maintaining cellular homeostasis and promoting organismal fitness. Rather than acting in isolation, organelles engage in dynamic crosstalk through membrane contact sites, metabolite exchange and signalling interplay. In recent years, organelles have been increasingly recognized as critical regulators of ageing and longevity. Here we summarize age-related organellar changes, highlight organelle-mediated intra- and intercellular signalling communication in lifespan and healthspan regulation, and discuss the active roles of organelles in microbiome-host interactions and transgenerational inheritance in regulating longevity. We further outline how longevity-promoting interventions influence organelles, and provide perspectives on how future technological advances may further accelerate progress in this emerging research topic.

Spontaneously blinking fluorophores toggle between nonfluorescent and fluorescent forms without caging groups or redox buffers, enabling super-resolution imaging. The intrinsic blinking of such dyes is governed by molecular structure and modulated by environment; there is no one-size-fits-all fluorophore suitable for every imaging context. We report dyes with tuned on:off ratios that enable single-molecule localization microscopy and super-resolution optical fluctuation imaging of biomolecular structures in vitro and in cells.

Object recognition depends on the ability to extract stable representations across changes in how they are viewed, yet it remains unclear how this capacity depends on visual acuity and cortical hierarchy. We combined behavioral testing and computational modeling to determine whether tree shrews, close relatives of primates with lower spatial acuity, can perform transformation-tolerant object recognition. Front-end modeling incorporating species-specific optics and photoreceptor sampling showed that, when scaled for acuity, tree shrew retinal filtering preserves the similarity structure of natural image categories relevant for object recognition. Behaviorally, tree shrews reliably discriminated complex objects across variations in position, scale, and viewpoint, including when embedded within natural scenes, and generalized to novel exemplars. Their recognition behavior was best explained by visual features emphasizing differences in global shape and size between objects and by representations from intermediate and deep layers of hierarchical neural network models. These results demonstrate that visual processing supporting object-level generalization can arise within visual systems lacking high-acuity front-end optics and establish the tree shrew as a key model for understanding the computational and evolutionary origins of high-level vision.

The global microscopy community has made wide efforts in imaging technology dissemination, especially to lower- and middle-income countries. Yet, many efforts have not fully realised their aim of increased, sustained microscopy utilisation. To guide future outreach initiatives towards more positive results, we analysed over 2300 unique applications across seven recent international microscopy workshops. We found significant differences in research priorities, as well as in microscopy experience and applications between lower- and higher-income regions. We discuss the importance of tailoring technology dissemination, training curricula, and capacity-building to these regional variations.

There is strong evidence that synaptic plasticity is a critical cellular mechanism underlying learning and memory. Although the forms of synaptic plasticity used by different circuits and cell types vary, a widespread presumption is that the male and female brain has evolved to use the same form of plasticity within the same circuits during performance on the same task. Here, we used complimentary approaches to determine how activity in the mouse frontal cortex supports the extinction of associative memories in a context-dependent manner. While in vivo recordings show that both male and female mice have similar cue-relevant activity patterns and ensemble dynamics in excitatory neurons from the infralimbic cortex (IL) during learning, activity in amygdala-projecting IL neurons was indispensable for extinction memories only in male mice. Likewise, male but not female mice showed evidence for the recruitment of IL by structural remodeling and clustering of dendritic spines on these neurons, and extinction memory impairments were evident only in male mice after projection-specific IL deletion of the glutamate receptor subunit GRIN2B. This work provides strong evidence that synaptic plasticity mechanisms employed during learning and critical for memory retrieval differ between males and females, which undercuts the utility of one-size-fits all therapeutic approaches for mental health conditions in which memory is disrupted.Competing Interest StatementThe authors have declared no competing interest.

Light sheet fluorescence microscopy (LSFM) is increasingly appreciated as the gold standard for gentle, volumetric imaging with fast acquisition speeds and/or long imaging durations. However, the often-constrained sample space of these microscopes has precluded a specific class of biological specimens from being studied with these tools: those requiring an air-liquid interface (ALI). Here, we present a device for robust imaging at ALI on an upright light sheet microscope with dipping objectives. We demonstrate the system using three relevant use-cases: ex vivo embryonic mouse salivary glands, human epidermal equivalent cultures, and in vivo adult Drosophila melanogaster brains. While the device presented is engineered for one specific light sheet microscope design, it provides a blueprint for easy adaptation to other systems. In doing so, it can potentially spur the use of LSFM for model systems that have so far been unable to take advantage of this powerful technology.

At the blood-tissue interface, vasculature luminal surface is critical for molecular transport, signaling transduction, and cell extravasation. Here, we present a method for proteomic profiling of the vasculature luminal surface in vivo, broadly applicable to any vertebrate. Quantitative mass spectrometry revealed the luminal surface proteome of the mouse brain vasculature and its temporal evolution from development to aging. In vivo genetic perturbation found that the arginine transporter SLC7A1 and the nitric oxide synthase NOS3 are needed for blood-brain barrier integrity in neonatal but not adult mice, whereas the hyaluronan degradation enzyme HYAL2 safeguards the barrier throughout the lifespan. By characterizing the proteomic dynamics of the vasculature luminal surface, the study links the metabolism of nitric oxide and hyaluronan to blood-brain barrier integrity.

Isocitrate lyase 2 (ICL2) from Mycobacterium tuberculosis undergoes dramatic conformational rearrangements upon binding to the allosteric effector acetyl-CoA. Time-resolved cryo-EM captured conformational states along the ICL2 activation trajectory, revealing how acetyl-CoA binding at the allosteric sites leads to asymmetric, half-of-site activity at the catalytic centres. These findings support a conformational selection model of allostery, whereby acetyl-CoA binding shifts the pre-existing equilibrium towards an active state of the enzyme.

Connectomics has become essential for the study of brain function, yet for most research groups it remains prohibitively costly in imaging time, data storage, and analysis. Here, we present an imaging, processing, and analysis pipeline for multi-resolution image acquisition and circuit reconstruction. Applied to the central complex of six insect species, we were able to obtain global projectomes at cellular resolution (40-50 nm) with embedded local connectomes describing key computational compartments at synaptic resolution (8-12 nm). We provide standardized protocols for volume EM sample preparation, image acquisition and image alignment, combined with existing methods for µCT block trimming, automatic segmentation, synapse detection, collaborative skeleton tracing with CATMAID, and segmentation proofreading via CAVE. We validated our workflow by reconstructing head direction cells across all six insect species, which revealed deep conservation at the level of cell types, cell numbers and projection patterns, while also revealing circuit level specializations. Overall, our pipeline democratizes comparative connectomics by making this method accessible for small research groups with modest resources.