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.

Biological memory systems store single experiences while continuing to learn, but how one-shot plasticity limits interference with existing memories is unclear. Behavioral timescale synaptic plasticity (BTSP) rapidly modifies synapses active within seconds of a dendritic plateau. We isolate its plateau-triggered component in a model where plastic weights and a stable instructive pathway jointly determine whether a plateau occurs, closing a feedback loop between the synaptic state and the plastic event that modifies it. For unstructured inputs, instantiated by independent uniform signed patterns, the dynamics reduce exactly to pathway alignment, which determines the fidelity of the instructed representation, the synaptic-turnover rate and the mean rewrite interval. For structured inputs, instantiated by correlated bimodal Curie–Weiss patterns, the instructive pathway biases which component of input structure enters the plastic synaptic state. In a BTSP-inspired continual-recognition network, combined instructive and plastic drives determined the selected memory unit for each one-shot write, whereas a Hebbian control used the same plastic weights for credit assignment and memory storage. The BTSP-inspired network remained accurate at longer repeat lags than Hebbian controls, an advantage that grew with network size, with both architectures optimized independently at every repeat lag. A reduced theory predicted held-out accuracy, lag capacity and dynamics of memory-trace strength directly from optimized parameters. It showed why intermediate proximal and distal coupling was optimal: proximal plastic drive guided plateau generation toward selected memory units, slowing synaptic turnover but limiting new encoding, whereas distal instructive drive enhanced familiar responses but could also make novel inputs appear familiar. The memory-trace strength in the rate-and-depth-matched Hebbian control still decayed faster and showed less effective credit assignment than in the BTSP-inspired network. These results connect dendritic plateau physiology to continual memory and support partial separation of allocation from storage as a mechanism for limiting interference during continual learning.

The cell cycle is tightly regulated by checkpoint mechanisms that ensure faithful duplication and segregation of the genome. Here, we induced cell-cell fusion between mitotic and interphase cells to study how nuclei from different cell cycle stages behave in a shared cytoplasm. We found that mitosis is a dominant cell cycle state: the mitotic cytoplasm can drive interphase nuclei into mitosis, whereas, in high ratios of interphase versus mitotic nuclei, fusion forced mitotic nuclei to exit mitosis. Both outcomes represent checkpoint override events with impactful consequences. Interphase nuclei forced into mitosis form aberrant mitotic spindles, show partially condensed DNA and ultimately undergo mitotic catastrophe. Conversely, forced mitotic exit resulted in reformation of nuclear envelope membranes around condensed chromosomes, forming nuclei with a defective nuclear import machinery. Altogether, cell-cell fusion revealed the consequences of checkpoint override, forcing nuclei through untimely cell cycle transitions, and highlight how cell-cell fusion experiments can be a powerful system to study how competing cytoplasmic states are integrated in a shared cytoplasm, such as in muscle, placenta formation and in cancer.

An animal's ability to survive and thrive-whether fleeing from danger, eating a meal, or fighting an infection-arises from the collective moment-to-moment activity of many interacting cell types throughout the body. Physiology seeks to elucidate these cellular interactions that span organs, cell types and timescales, but has been limited by the inability to record this time-varying cellular activity simultaneously throughout the entire body. Here we develop WHOLISTIC (WHole-Organism Live-Imaging System for recording Tissue and IntraCellular activity), a method to image second-timescale activity of cells across the entire vertebrate body at cellular resolution. WHOLISTIC advances and integrates volumetric fluorescence microscopy, machine learning, and pancellular transgenic expression of calcium sensors1, demonstrated in larval zebrafish, with proof of concept in adult Danionella cerebrum. To access information about the molecular and ultrastructural substrates for the measured dynamics, we advanced whole-body expansion microscopy2. At the cellular scale, body-wide screening revealed unexpected responses, including chondrocyte reactions to cold and meningeal responses to ketamine. At the organ scale, WHOLISTIC identified rhythmic travelling waves along the renal nephron. At the multi-organ scale, it revealed unknown muscle synergies and muscle-organ interactions. At the whole-organism scale, the method captured brainstem-controlled redistribution of body-wide blood flow. Combining optogenetics with WHOLISTIC enabled all-optical causal dissection of brain-body interactions. These advances establish a paradigm for systems biology that bridges cellular and organismal physiology, enabling comprehensive discovery across scales-from fundamental mechanisms to therapeutic targets.

Robust homeostatic plasticity is essential for survival, enabling neuronal circuits to withstand destabilizing forces and restore function during critical challenges such as predator evasion or toxin exposure. Semaphorin/Plexin signaling is central to presynaptic homeostatic potentiation at central synapses and at neuromuscular junctions across species. However, our understanding of this pathway has remained incomplete, as secreted Semaphorins bind weakly to their Plexin receptors and require additional coreceptors for efficient signaling. Here, we identify Neuropilin and Tolloid-like protein (Neto-α), an auxiliary subunit for ionotropic glutamate receptors (iGluRs), and the tyrosine kinase Abelson (Abl) as essential components of the Sema2b/PlexB signaling pathway that drives rapid homeostatic potentiation and stabilizes synaptic strength at the neuromuscular junction. Neto-α functions as a Neuropilin-like coreceptor that cooperates with Sema2b to relieve PlexB autoinhibition and initiate signaling. In parallel, Neto-α recruits Abl, which functions as a cytosolic amplifier to enhance pathway output. We demonstrate that both pathway activation and amplification are required for a rapid and effective homeostatic response. By integrating these two functions within a single molecular assembly, Neto-α ensures fast and efficient compensatory responses to perturbations. This evolutionarily conserved signaling module, Neto (or Neuropilin)/Sema/Plex/Abl, likely operates in cellular contexts beyond neural function, including in tumorigenesis.

Centromeres, defined by CENP-A-containing nucleosomes, direct the assembly of kinetochores for spindle attachment. In mitosis, CENP-A and the constitutive centromere-associated network (CCAN) of the inner kinetochore are arranged into bipartite subdomains within clearings of chromatin. However, it remains unclear whether any of these features exist before mitosis. We show that in interphase, CENP-A and the CCAN assemble \~200-300 nm shell-like structures that enclose a chromatin-poor central cavity. Strikingly, this cavity is occupied by the interphase-specific CENP-A chaperone complex, which promotes CENP-A assembly once per cell cycle. However, chaperone presence, but not CENP-A incorporation, is required to generate both the shell architecture and the chromatin clearing. The CCAN scaffold CENP-C, which links CENP-A nucleosomes to the chaperone complex, exhibits radial organization spanning the entire structure and is essential for its formation. These data uncover a previously unrecognized structural role for the CENP-A chaperone machinery in establishing interphase centromere architecture and suggest a mechanism by which this machinery configures centromeres for faithful kinetochore assembly and genome stability.

In the wild, a mouse must flexibly perform multiple computations at once, rapidly navigating, sampling its environment, forming and using memories, and balancing internal needs. Capturing the self-paced, low-repetition, complex nature of decision making in the wild while preserving the experimental control needed to interpret its process remains a challenge. Here we show that lab mice, without food restriction, learn to forage for hidden resources cued by ambiguous sounds in a dynamic environment by spatiotemporally reorganizing their existing behavioral repertoire. Using a novel closed-loop assay, we required mice to sit still to receive informative but hard-to-localize sound cues emanating from one correct location they must find, out of 157 possible locations in a large arena. With experience, mice become efficient hunters. Efficient hunting cannot be explained by an increase in a particular behavioral module, but instead, mice reorganize existing behaviors into clustered bouts of high-quality sampling and site-checking. Looking forward, this work establishes an approach for studying the neural, molecular, and evolutionary basis of naturalistic decision-making in mice.

Whether recovering after a gust of wind, or rapidly saccading away from an oncoming predator, fruit flies show remarkable aerial dexterity about their body roll axis. Here, we investigated the detailed wing kinematic changes during free-flight roll motion and probed the neuromuscular basis for such changes. Consistent with previous work, we observed that flies manipulated the stroke amplitude difference between their wings to control their roll angle. Here, we show that flies are capable of achieving such changes by altering the stroke amplitude of either or both of their wings. Further we found that during corrections flies can also take advantage of an aerodynamically significant change in the angle of attack of their uppermost wing. Curiously, these corrective wing changes cannot be eliminated when motor neurons hypothesized to be used during roll maneuvers (i1, i2, b1, b2, and b3) are individually inhibited. However, free-flight optogenetic manipulations and quasi-steady aerodynamic calculations show that each of these motor neurons individually can effect kinematic changes consistent with a roll correction. Combining this evidence with an analysis of haltere inputs found in the BANC connectome, we propose that the observed robustness could be the result of two sets of muscular redundancies that receive shared inputs from haltere sensory afferents: one set, containing b1 and b2, is able to increase the stroke amplitude of the lower wing; while the other set, containing i1, i2, and b3, is able to decrease the stroke amplitude and wing pitch angle of the upper wing. Because of the redundancy in the input sensory information and output wing motion in the muscles in each cluster, the fly is able to perform roll stability maneuvers even when one of the constituent motor neurons is inhibited. This framework proposes new ways fast aerial maneuverability can be implemented when dealing with the fly’s most unstable rotational degree of freedom.

Yeast two hybrid (Y2H) assays serve as powerful platforms for the detection and engineering of protein-protein interactions. Here, we describe a strategy for using Y2H-based screening to assess and develop optogenetic tools based on light-controlled protein-protein interactions.