Two-photon calcium imaging enables measurements of neural activity from individual neurons and their subcellular processes in awake animals. Axial brain motion can change measured fluorescence independently of neural activity and move small structures out of the imaging plane, confounding two-photon calcium measurements in behaving animals. We developed two complementary post-acquisition methods, selected according to whether the imaged structure remains detectable across the encountered depth range. When it does, z-stack correction estimates imaging depth from a separately acquired reference z-stack and uses an axial fluorescence profile specific to each neural structure to rescale fluorescence to a common reference depth. When a structure can leave a single imaging plane, volume-based z-registration reconstructs a common-depth movie from repeated acquisitions of closely spaced planes. In awake mice, both methods tracked experimentally imposed axial displacements within the sampled range, with depth-estimation root mean squared errors (RMSEs) below 1 μm for z-stack correction and 0.69-1.03 μm for volume-based z-registration. Correction changed fluorescence substantially for a subset of somata, with larger effects in axonal boutons. Both methods are implemented in suite2p-in-depth, a Python package that integrates with Suite2p and provides diagnostics for depth estimation, correction quality and data reliability. Together, these methods enable axial-motion artifacts to be assessed and corrected within established Suite2p workflows.

Understanding the spatial organization of biomolecules in living organisms is a central but challenging goal in biology. Here, we introduce peroxidase reactions activated by D-amino acids (PRADA), which uses an engineered oxidase to convert nonproteinogenic D-amino acids into HO for in situ activation of a genetically fused peroxidase. PRADA enables versatile proximity-based chemistries, including protein labeling, RNA labeling and functional polymer assembly, with minimal toxicity and background. We demonstrate that PRADA is broadly applicable inside living organisms, including flies, worms, fish and mice. By further leveraging the unique RNA reactivity of PRADA, we developed a mutational profiling sequencing strategy for spatiotemporally resolved mapping of RNA secondary structure. Application of this multifaceted PRADA platform in a mouse xenograft model offered insights into its mitochondrial proteome, transcriptome and RNA structurome, revealing RNA folding as a regulatory mechanism in mitochondrial gene expression. Collectively, PRADA holds promise to uncover the intricate organization of biomolecules in vivo.

Sarcomeres, the basic repeating unit of striated muscle, are joined together by crosslinked actin filaments found at the boundaries of muscle sarcomeres, termed Z-discs. Z-discs play a key role in cardiac signalling and disease, however, the arrangement and function of many of the proteins present in the Z-disc remain to be understood. Here, we determined the organisation of 3 key proteins, ZASP, ɑ-actinin-2 and the Z1Z2 epitope of titin, located within the Z-disc. We fluorescently labelled these proteins in cardiac myofibrils using Adhirons specific to each protein and used interferometric photoactivated localization microscopy (iPALM) to obtain the 3D position of these proteins to a high precision (<10nm in x,y,z). We then used PERPL (Pattern Extraction from Relative Positions of Localisations: available at https://github.com/oubino/z_disk/releases/tag/v0.0.3) to analyse patterns in the relative positions of the proteins and reveal their underlying organisation. This analysis revealed that ZASP and ɑ-actinin-2 have a similar repeating organisation, but that the organisation of Z1Z2 is different.

Neural activity is metabolically costly and supported by local increases in cerebral blood flow. The process by which neural activity drives blood flow increase, neurovascular coupling (NVC), remains poorly understood. One persistent knowledge gap is how subcellular compartments are arranged at the vascular wall, where local signaling occurs. This requires a survey of the neurovascular interface at the nanometer scale. To this end, we analyzed a ∼1 mm3 volume of mouse visual cortex imaged with serial electron microscopy. We manually labeled different vascular zones and classified the EM segments surrounding hundreds of vessel segments across these zones. We found that capillaries and venous vessels are surrounded by more axonal volume than arteriolar vessels. Perivascular axons tended to bundle into micro-tracts, where axons coursed near the vessel wall in a variety of geometric orientations. Micro-tracts covered ∼60% of the surface of capillaries, but only 24 – 40% of every other vascular zone’s surface. Finally, we show that the basket cells, with their extensive axonal arbors, are candidate members of micro-tracts: on average, their axons approach 2-to-3-fold more capillaries than other neuronal subtypes. Altogether, perivascular axonal micro-tracts may be an important physical substrate for NVC signaling at capillaries.

Archaeal histone-based chromatin complexes are the evolutionary precursor of the eukaryotic nucleosomal landscape. Histones are encoded by most Archaea and are present in sufficient quantities in many heat-loving species to plausibly completely wrap and condense the genome(s). Establishing whether the in vivo archaeal histone-based chromatin structure(s) align to the repeating extended polymeric structures revealed with purified components in vitro remains an important goal to link the biological roles of archaeal and eukaryotic histone-based chromatin structures. Deploying advanced imaging and cryo-electron tomography techniques with the hyperthermophilic, histone-encoding archaeon Thermococcus kodakarensis, we reveal that the in vivo histone-based chromatin architecture changes dramatically during the growth cycle. While the cytoplasm contains a relatively uniform nucleoid in exponential growth, tightly packed, helical filaments that conform to archaeal histone-based chromatin observed in vitro emerge during the transition to stationary phase. The in vitro extended chromatin conformation of histone-based genome architecture observed in vivo establishes that archaeal information processing machinery can function on tightly packed and regularly organized histone-based chromatin structures. We also observe large, nucleic-acid rich, globular condensates directly alongside the helical filamentous chromatin conformation that may represent an additional conformation of archaeal chromatin. Our results suggest that alternative forms of archaeal chromatin may be achieved without obvious chromatin remodeling complexes or biologically significant levels of post-translational modifications to histone proteins, providing a plausible evolutionary driver for the emergence of more complex mechanisms that protect and regulate expression of eukaryotic genomes.

High-spatial-resolution in situ mapping of biomolecules within tissue reveals critical insights into the complex molecular landscape and spatial organization of biological systems. Mass spectrometry imaging (MSI) is a powerful tool for spatially resolved molecular analysis of biological samples, with ongoing demand for improved spatial resolution. Tissue expansion combined with MSI (TEMI) is a recently developed approach that enables multiomics molecular mapping across various biological tissues with significantly improved spatial resolution. Unlike conventional methods that depend on instrument-based enhancements in spatial resolution, TEMI physically enlarges tissue samples via harsh-condition-free hydrogel expansion, achieving more than 3.5-fold increase in effective imaging resolution using standard MSI instrumentation. TEMI delivers single-cell spatial resolution in tissue samples and enables detection of biomolecular heterogeneity that remains uncharacterizable in unexpanded tissue using conventional MSI. Notably, TEMI supports high-spatial-resolution mapping of multiple biomolecular classes-including lipids, metabolites, N-glycans, peptides and proteins-within a single tissue sample. Here, we provide a detailed, step-by-step guide for TEMI, including hydrogel-based tissue expansion under mild conditions, cryosectioning of the expanded tissue-hydrogel sample, a comprehensive experimental workflow for multiomics TEMI on a single tissue section, data acquisition and visualization pipelines, as well as troubleshooting tips. Overall, we demonstrate that TEMI overcomes the long-standing spatial limitations of MSI without requiring hardware modifications, ensuring compatibility with existing MSI instruments and promoting broad accessibility and adoption within the research community.

The gold standard datasets for mapping connectomes are electron microscopy volumes of densely labeled neural tissue at nanometer resolution. Yet reconstructing and proofreading neuronal arbors and annotating all synapses requires pipelining multiple software tools that are often fragmented, inconsistently maintained, or proprietary, hindering reproducibility and automation. Here, we introduce Catena, an open-source, comprehensive, developer-centric software suite for connectomics that integrates modules for 3D neuron and organelle segmentation, synapse detection, microtubule tracking, and neurotransmitter inference. Catena organizes its modules in composable, chunk-wise processing pipelines in a completely documented, extensible, and adaptable design. We further reduce compute and ground-truth data requirements with pretrained machine learning models, facilitating fine-tuning. Catena ships fully containerized modules that encapsulate evolving dependencies for consistent execution across workstations and clusters. By consolidating open components, shareable models, and containerized runtimes, Catena delivers a reproducible and scalable approach to mapping cellular connectomes from electron microscopy volumes. Code and documentation: this https URL

Cell-state diversity drives tissue adaptability, repair, and disease resilience, but capturing this complexity is a challenge. Current approaches rely on transcriptional profiling and overlook organelle structure, a key indicator of metabolism and stress. We developed spatial Organellomics (sOrganellomics), an imaging workflow that integrates automated segmentation with machine learning to classify and spatially map cell states from multi-organelle signatures. In liver and pancreas, these signatures distinguished broad cellular classes. In liver, sOrganellomics revealed that zonal position did not fully explain organelle-defined hepatocyte categories. Instead, hepatocytes formed intermixed communities within canonical zones, supporting a refined subzonal diversity model. Nutritional stress reshaped this organization. Intravital imaging linked fasting-induced organelle remodeling with altered mitochondrial membrane potential in vivo, supporting multi-organelle architecture as a structural readout of tissue adaptation.

Fluorescence microscopy applications often require specialized instruments that are optimized for different experimental goals. Here, we present a reconfigurable microscopy module that integrates highly inclined swept tile (HIST) illumination for high-sensitivity single-molecule imaging and line-scanning confocal microscopy for rapid and optically sectioned volumetric acquisition. The system shares major hardware components, including lasers, scanning optics, and detection hardware, while employing unique beam shaping pathways to enable rapid switching between modalities without realignment. We characterize the module performance by measuring the excitation beam profiles, the point spread functions (PSF), and the optical transfer functions (OTF) across 40x, 60x, and 100x magnifications and demonstrate imaging applications including diffraction-limited fixed and live-cell volumetric imaging, fluorescence recovery after photobleaching, and super-resolution DNA-PAINT and single particle tracking (SPT). We also demonstrate the capability to execute multimodal imaging workflows by performing confocal imaging for chromatin density classification correlated with SPT data of nuclear proteins with diverse functions. Together, these results demonstrate a versatile imaging platform capable of supporting complementary fluorescence imaging modalities within a single instrument.

Nervous systems form wiring patterns that are reproducible across individuals. This reproducibility is thought to emerge from molecular encoding and developmental events, but their relative contributions remain unclear. We address this question in the C. elegans neuropil, where embryonic developmental dynamics and adult anatomy are resolved at single-cell resolution. We find that transient morphogenetic structures — rosettes, corridor cells, pioneer axon scaffold — restrict which axons make contact, shaping the neuropil into overlapping neighborhoods. This demonstrates how early events constrain wiring choices, but not whether they explain the resulting reproducibility. To explain, we use an agent-based model of stochastic innervation that recapitulates macro- and micro-level reproducibility, revealing a trade-off between physical constraint and molecular specificity that limits neighborhood size. Counterintuitively, less selective axons produce more reproducible wiring when constrained within neighborhoods. This trade-off lets nervous systems maximize reproducibility without having to molecularly encode every axon-contact, a strategy for robust yet flexible wiring.