Summary Fluorescent imaging in live cells is a cornerstone of life sciences. While natural fluorescent proteins have been engineered to enhance individual features, no existing tag combines ideal properties into a single system: high brightness, reversible binding, compact size, and stability across diverse conditions. Here, we achieve this through de novo design of rhodamine binders (Rhobin). To harness the broad repertoire of rhodamine fluorophores, we developed a generalizable design strategy for a pan-rhodamine binder compatible with diverse wavelengths and applications. Rhobin enables live- and fixed-cell imaging of various subcellular targets in mammalian cells, showing brightness surpassing existing tags. Its reversible fluorophore binding supports super-resolution stimulated emission depletion (STED) and live-cell single-molecule imaging for extended durations compared with HaloTag. Beyond conventional systems, Rhobin enables live imaging of the extremophile Sulfolobus acidocaldarius at 75°C, previously inaccessible with current tags. Together, these results establish Rhobin as a versatile platform for next-generation imaging and biosensor design.

To select behaviors appropriate to their circumstances and needs, animals integrate information about the external environment with information about the state of their bodies, derived from sensing and processing internal signals (interoception). However, the brain-wide circuit activity underlying interoception and its integration with sensorimotor processing remains unclear, partly because of technical barriers to accessing whole-brain activity at the cellular level during physiological perturbations. We developed an all-optical system for whole-brain neuronal imaging in behaving larval zebrafish during optical uncaging of gut- or bloodstream-targeted nutrients and visuo-motor stimulation. Widespread neural activity throughout the brain encoded nutrient delivery, unfolding on multiple timescales across many peripheral and central regions. Evoked activity depended on delivery location and occurred in response to both amino acids and D-glucose, but not L-glucose. Many gut responsive neurons also responded to swimming and visual stimuli, with brainstem areas primarily integrating gut and motor signals and midbrain regions integrating jointly gut, visual, and motor signals. This platform links body-brain communication studies to brain-wide neural computation in awake, behaving vertebrates.

Expansion microscopy (ExM) enables nanoscale fluorescence imaging on standard microscopes, but its combination with single-molecule localization microscopy (SMLM) remains challenging due to the incompatibility of expanded hydrogels with photoswitching buffers. Here, we introduce a single-step expansion microscopy approach that enables SMLM using spontaneously blinking dyes in 6–14× expanded samples, without re-embedding or buffer exchange. Using this approach, we achieve nanometer-scale spatial resolution by resolving the organization of the nuclear pore complex and the molecular structure of recombinant homotrimeric proliferating cell nuclear antigen.

Cells depend on the spatial organization of proteins, RNA, and DNA into discrete subcellular compartments. Previous methods have largely centered on measuring spatial organization based on only one of these biomolecular classes at a time. Here, we demonstrate that POCA photocatalytic proximity labeling can serve as a unified photosensitizer-based platform for profiling the proximal proteomes of protein, RNA, and DNA targets within a single experimental framework. We show that POCA can harness standard immunofluorescence or in situ hybridization workflows to specifically target organic fluorophore photosensitizers to intracellular targets for proximity labeling in fixed cells. POCA-targeted proximity labeling requires minimal cellular input and does not require genetic engineering. Additionally, POCA photosensitizers are selected to also be fluorescent, enabling direct confirmation of on-target localization by imaging prior to proteomic analysis. To demonstrate broad utility, we apply POCA across multiple molecular targets spanning protein, RNA, and genomic DNA, including components of the nuclear pore complex, nucleolus, nuclear speckles, telomeres, and pericentromeric heterochromatin. By anchoring proximity labeling to both a protein and an RNA within the same nuclear compartment, we resolve shared and distinct proximal proteomes from orthogonal molecular perspectives.Competing Interest StatementD.K.S. is a collaborator with Thermo Fisher Scientific, Genentech, Calico Labs, Matchpoint Therapeutics, and AI Proteins. K.M.B is a collaborator with Thermo Fisher Scientific and on the advisory board for Matchpoint Therapeutics. B.J.B. has filed a patent application covering aspects of this work (US Patent App. 18/728,937). B.J.B. is listed as an inventor on patent applications related to the SABER technology related to this work (US Patent 11,492,661; US Patent App. 18/607,269). E.L.H. also collaborates with Thermo Fisher Scientific, Genentech, and Xaira Therapeutics and consults for Calico Labs, Matchpoint Therapeutics, and Flagship Pioneering. Patents and patent applications covering azetidine-containing rhodamine dyes (with inventors J.B.G. and L.D.L.) are assigned to HHMI. L.D.L. is a scientific cofounder, consultant, and shareholder of Eikon Therapeutics. The other authors declare no conflicts.National Institutes of Health, R35GM137916, R35GM150919, DP2GM146246, P30 CA015704, U24HG006673, T32HL007093W. M. Keck Foundation, https://ror.org/000dswa46Pew Charitable Trusts, https://ror.org/02xhk2825Andy Hill CARE FoundationDavid and Lucile Packard Foundation, https://ror.org/032atxq54Damon Runyon Cancer Research Foundation, https://ror.org/01gd7b947

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.

Proteins essential for signaling, morphogenesis, and migration traverse the complex intracellular landscape via vesicular trafficking, microtubule-based transport, and diffusion. However, the precise mechanisms guiding soluble proteins toward their functional destinations have remained elusive. Here, we demonstrate that soluble proteins are directed toward the cell's advancing edge through advection-diffusion enhanced by intracellular fluid flow. We reveal that advective transport occurs within a specialized compartment at the cell's leading edge, separated from the rest of the cytoplasm by an actin-myosin condensate barrier. The barrier limits protein mixing between the compartment and the rest of the cytoplasm, maintaining localized protein concentrations. Contraction at the barrier generates a molecularly non-specific fluid flow that drives the forward movement of treadmilling actin monomers, actin-binding proteins, adhesion molecules, and even inert proteins. Dynamic changes in the local curvature of the barrier steer the fluid flow to direct proteins toward protrusive regions of the leading edge. This advective mechanism synchronizes protein distribution with local changes in cell morphology. Outside this compartment, diffusion dominates as the principal mode of soluble protein transport. Our findings uncover previously unrecognized compartmentalization strategies that regulate soluble protein concentrations and coordinate their efficient distribution for homeostasis, protrusion, and adhesion.

Mitochondria utilize calcium to increase ATP synthesis. However, excessive matrix calcium activates the mitochondrial permeability transition (mPT), a process that permeabilizes the mitochondrial inner membrane and leads to cell death. While initially characterized 50 y ago, the proteins underlying the process are unclear, although integral membrane proteins were expected to be the porous entities during calcium overload. Here, we designed two assays to study the mPT using high-throughput methodologies. By surveying 19,113 proteins in human cells, we identified four proteins that sensitize the human mPT, but only one that was essential for mPT activation, mitochondrial-localized NRLX1. Surprisingly, NLRX1 is not an integral membrane protein, and our work did not identify any essential integral membrane proteins for the human mPT. The mitochondrial permeability transition (mPT) is an evolutionarily conserved destructive process that permeabilizes the inner mitochondrial membrane in response to calcium overload. The molecular mechanism underlying the mPT is not established. To unambiguously identify essential proteins, we designed two phenotypic assays for mitochondrial calcium overload and applied them to FACS-based CRISPR screening in human cells, ultimately evaluating 19,113 genes. The first screen studied mitochondrial membrane potential (MMP) collapse in response to calcium overload. Top-ranked genes were the essential proteins of the mitochondrial calcium uniporter complex, MCU and EMRE, reflecting that the calcium-induced MMP collapse results from mitochondrial calcium entry and not the mPT. The second screen measured the permeability of the inner mitochondrial membrane. Here, the fluorescent interaction of a membrane impermeant 600 Da dye and a mitochondrial-targeted HaloTag protein was studied under mPT activating conditions; calcium overload and the thiol-reactive molecule phenylarsine oxide. With secondary validation, we identified four protein-encoding genes that delayed or prevented the mPT under knockout: NF2, REST, BPTF, and NRLX1. Knockout of the nonmitochondrial proteins BPTF, NF2, or REST increased mitochondrial calcium retention capacity (CRC). However, calcium release or sensitivity to cyclosporin A (CsA) persisted, indicative of mPT sensitizers. Only knockout of the mitochondrial matrix protein, NLRX1, increased CRC, abolished calcium release, and was CsA-insensitive. This top-ranked hit of the mitochondrial permeability screen meets the definition of an essential mPT activator. Integral membrane proteins, including all previously proposed mPT candidates, were not essential activators.

Expansion microscopy (ExM) enables nanoscale imaging on standard microscopes, but combining ExM with single-molecule localization microscopy (SMLM) remains difficult, owing to the incompatibility of expanded hydrogels with photoswitching buffers. Here, we introduce a single-step expansion microscopy method that allows SMLM with spontaneously blinking dyes in 6-14× expanded samples, without re-embedding. We demonstrate nanometer-resolution imaging by resolving the organization of the nuclear pore complex (NPC) and the molecular structure of recombinant homotrimeric proliferating cell nuclear antigen (PCNA).

Fluorescence microscopy is a powerful tool for studying biomolecules in their native environments, offering high spatio-temporal resolution but requiring fluorescent labels. Current live-cell compatible labeling strategies repurpose natural systems, such as fluorescent proteins or proteins that bind fluorescent ligands. While advances have been made to engineer natural proteins into labels with minimal size, high brightness, as well as enhanced thermo- and photostability, these approaches often require trade-offs among desirable properties due to the inherent limitations of re-engineering natural proteins. In this work, we present rhodamine binder (Rhobin) tags—de novo-designed proteins that bind rhodamine-derived fluorophores. Unlike traditional approaches, Rhobin tags were developed by directly incorporating desirable features during the computational design process, which resulted in compact binders with outstanding thermostability. Their nanomolar substrate affinity, rapid labeling kinetics, and orthogonality to established labeling systems such as HaloTag and SNAP-tag facilitate versatile live and fixed-cell imaging of diverse subcellular targets in mammalian cells. Transient fluorophore binding further enables advanced imaging techniques, including live-cell super-resolution STED microscopy with reduced photobleaching and single-molecule localization microscopy in live and fixed cells. To the stability of Rhobin tags under extreme environmental conditions, we demonstrate showcase protein tagging and timelapse imaging in the extremophile Sulfolobus acidocaldarius living at 75°C, an application previously inaccessible with existing tags. We anticipate that Rhobin tags will become a central component in the toolbox of fluorescent labels and will pave the way for a new generation of modular protein tags and biosensors with tailor-made properties.

Dendrites transform local electrical activity into intracellular Ca2+ signals that drive plasticity1,2, yet the voltage→Ca2+ mapping during natural behavior remains poorly defined. Here, we measure this transfer function via simultaneous voltage and Ca2+ imaging throughout the dendritic arbors of hippocampal CA2 pyramidal neurons in behaving mice. Dendritic Ca2+ exhibited a hierarchical activation pattern dominated by back-propagating action potentials: simple spikes primarily drove somatic and proximal Ca2+, whereas complex spikes produced larger somatic Ca2+ signals and propagated farther into distal dendrites, sometimes in a branch-selective manner. Dendrite-restricted co-activation of voltage and Ca2+ without concurrent somatic events was rare. A biophysics-inspired model accurately predicted local Ca2+ transients from local voltage waveforms. Our data and model provide a quantitative understanding of when – and why – dendritic Ca2+ signals in CA2 pyramidal cells arise during behavior.