Biophysical Reports

Temperature is a fundamental parameter governing all molecular processes, including those that define life. Fluorescence microscopy is a powerful tool to observe molecular processes in living systems in real time. Precise control and measurement of temperature during fluorescence microscopy is therefore essential. We present here a robust temperature measurement based on the excited-state lifetime of the widely available and relatively inexpensive fluorescent dye pentamethine cyanine (Cy5). The excited-state lifetime of Cy5 shows a monotonic decline in the measurement range of 0 {degrees}C - 80 {degrees}C. The measured dependency is linear until 39 {degrees}C and monoexponential above. The dependance of excited-state lifetime upon temperature is used to measure temperature up to a precision of 0.5 {degrees}C or less, a temporal resolution down to <1 millisecond and to resolve temperature gradients with spatial resolutions that are only diffraction-limited. The far-red excitation and emission of Cy5 leaves bandwidth to simultaneously measure at least 3 additional spectral channels in standard fluorescent microscopes simultaneously. We demonstrate determination of temperature during 4-color live-cell fluorescence microscopy for a temperature-controlled experiment. We also show its applicability in measuring temperature gradients and laser-induced sample heating such as during STED nanoscopy.

Protein oligomerization equilibria are central to many biological processes and are often highly sensitive to environmental conditions such as ionic strength, pH, and ligand binding. Quantitative characterization of these equilibria remains experimentally challenging because stable protein complexes frequently dissociate only at concentrations that are difficult to access with conventional biophysical methods. Fluorescence correlation spectroscopy (FCS) is uniquely suited to this problem, as it provides direct access to diffusion coefficients of fluorescently labeled proteins at nanomolar concentrations. However, the quantitative interpretation of FCS data from oligomeric systems requires a rigorous mathematical framework and careful experimental practice that have not previously been described in sufficient detail to guide implementation. Here, we provide a comprehensive description of the experimental workflow and analytical framework for determining dissociation equilibrium constants by FCS, covering instrument calibration, sample preparation, data quality control, after-pulse correction, and nonlinear least-squares fitting. We discuss common sources of error and provide practical guidance on critical experimental considerations including surface passivation, buffer preparation, equilibration time, and the role of labeling efficiency. Using the homotrimeric sliding clamp PCNA as a model system, we demonstrate the complete workflow under a range of KCl concentrations and show that moderate ionic strength stabilizes the PCNA trimer while very high salt partially destabilizes the complex. The approach is general and applicable to any reversible protein self-association reaction accessible by fluorescence detection at low protein concentrations.

Fluorescent dyes are critical to visualizing nucleic acids in many applications. SYTOX Orange and SYTOX Green are cyanine dyes, used in dead cell staining and increasingly in single-molecule assays to probe DNA supercoiling and processing. However, their structures and effects on DNA mechanics are not or only partially known. We determine the structure of SYTOX Orange to be (E)-2-((2-(4 ((diethyl(methyl)ammonio)methyl)phenyl)-6-methoxy-1-methylquinolin-4(1H)-ylidene)methyl)-4-methyloxazolo[4,5-b]pyridin-4-ium, identical to SYBR Gold except for an aza-benzoxazol core that is fundamentally different from other dyes in the SYTOX and SYBR families. We report SYTOX Green to be (Z)-2-(bis(3-(trimethylammonio)propyl)amino)-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-1-phenylquinolin-1-ium, similar to PicoGreen. Using magnetic tweezers, we characterize the effect of SYTOX Orange and SYTOX Green on DNA mechanics. They lengthen and unwind DNA consistent with intercalation and the DNA unwinding angles per dye are 21.1(1) degree and 20.5(1) degree for SYTOX Orange and Green, respectively. Both dyes leave the DNA bending persistence length and plectoneme size almost unaltered (<10% change up to 1 uM), which is advantageous in assays probing DNA supercoiling. Their photophysical properties reveal close agreement between single-molecule manipulation and optical absorbance and fluorescence spectroscopy. Our comprehensive set of complementary measurements relates mechanical and optical properties to the molecular structures and provides recommendations for their use in applications.

Mechanotransduction via the actin cytoskeleton is linked to fundamental cellular processes such as morphogenesis, cell division, and motility, requiring the control of tensile forces mediated by the motor protein non-muscle myosin 2 (NM2). Formins such as mDia1 have been shown to elongate actin structures that are under mechanical tension; conversely, mDia1s elongation rates are modulated by the applied force. Despite their relevance at the membrane/cortex interface, reported values for tension in formin-elongated actin filaments stem from theoretical estimates and simulations, but have not been amenable experimentally so far. Thus, we developed a Forster resonance energy transfer (FRET)-based, tension-sensitive probe (mDia1TS) and quantified the measured tension in live U2OS cells using fluorescence lifetime imaging microscopy (FLIM). Through whole-cell ROI analysis we show a short and long lifetime component, reporting an intensity-weighted, averaged lifetime corresponding to [~]3.5 pN. Upon mitogen stimulation of cells using EGF, we show that the tension homeostasis changed significantly, with a measurable increase in tension in the cells periphery and relaxation in its center. Furthermore, the reported average tension relaxed by 2 pN after adding the NM2 inhibitor para-nitroblebbistatin. We utilized siRNA knockdowns of individual NM2 paralogs (NM2-A, NM2-B, or NM2-C) to measure their individual contribution, revealing NM2-A as the main paralog to produce tensile force in this system. Taken together, we demonstrate that mDia1TS is able to directly determine that active mDia1 in cells is under tension, and that subcellular quantification with pN precision is possible. SignificanceDespite the fundamental importance of formins in regulating actin-based processes, reported values for tension in formin-mediated actin structures stem from simulations and theoretical estimates. In this study we developed a FRET-based, tension-sensitive reporter probe for formin mDia1, which we termed mDia1TS. Given the expanding clinical spectrum of DIAPH1/mDia1 mutations, our tool mDia1TS provides a quantitative tool for elucidation of changes in cytoskeletal assemblies.

Small-angle X-ray or neutron scattering (SAXS/SANS) analysis of large unilamellar vesicles (LUVs) is often limited by high-dimensional bilayer models and the lack of dedicated, statistically rigorous workflows. Here, we introduce SAS_MoCa, an open-source Python package that integrates a compositional scattering density profile (SDP) description of lipid bilayers with a separated form factor (SFF) treatment of vesicle size and polydispersity, and couples these highly parameterized models to an adaptive thermodynamic simulated annealing algorithm formulated within a constrained Bayesian framework. SAS_MoCa enables users to incorporate quantitative prior information from, e.g., previous SAXS/SANS studies, dynamic light scattering, NMR, or molecular simulations, and returns full posterior parameter distributions, uncertainties (reported as medians and median absolute deviations) and correlations even from single SAXS curves. Validation on POPC, POPE and DMPC SAXS-only data demonstrates that the method yields reproducible structural parameters with uncertainties comparable to joint SAXS/contrast-variation SANS analyses. The modular architecture of SAS_MoCa facilitates extension to additional lipid systems and future joint SAXS/SANS or SANS-only applications.

Integral membrane proteins remodel the surrounding lipid bilayer, but quantifying the resulting deformations and linking them to protein density in the membrane has remained challenging. Here, we introduce an integrative methodology that combines all-atom molecular dynamics (MD) simulations with multiscale small-angle X-ray scattering (SAXS) analysis to connect membrane strain to the protein/lipid ratio in proteoliposomes. Using outer membrane phospholipase A (OmpLA) reconstituted into lipid bilayers with both increased and decreased hydrophobic thickness, we systematically probe the effects of positive and negative hydrophobic mismatch.MD simulations demonstrate that OmpLA causes anisotropic, oscillatory thickness deformations extending up to eight times the radius of the first lipid shell surrounding the protein, yet the net change in average membrane thickness remains below 1%. Through our multiscale SAXS analysis, we quantitatively extract structural parameters, ranging from proteoliposome size to internal membrane architecture, using constrained Bayesian inference, with priors derived from MD findings. Specifically, we determine the protein/lipid molar ratio and average membrane strain, revealing excellent agreement between experiment and simulation. In thinner bilayers, substantial protein loss limits the analysis, highlighting the role of bilayer stability in sample preparation. Moreover, the predominance of OmpLA monomers in the thicker membranes is consistent with weak, membrane-mediated repulsive interactions between protein inclusions. Collectively, this integrative approach establishes a framework for quantifying protein-lipid interactions across molecular and mesoscale dimensions.

Single-particle tracking (SPT) over time enables direct observation of molecular transport and interactions in living cells. Fluorescence-based SPT has provided insights into intracellular processes such as endocytosis, receptor signaling, and drug delivery. Extending the observation window to several hours and beyond is critical for capturing slow intracellular dynamics, including the full course of endosomal trafficking, the long-term accumulation of particles within subcellular compartments, and transitions between transport modes that occur on hour-scale timescales. However, long-term intracellular SPT under visible-wavelength excitation remains challenging because fluorescence probes generally suffer from photobleaching and phototoxicity. While near-infrared (NIR) excitation can simultaneously mitigate these issues, generally weak emission of NIR-emitting dyes has hindered its wide application in long-term SPT. Here, we demonstrate long-term NIR SPT using atomically precise gold quantum needles, Au42(PET)32 (PET = 2-phenylethanethiolate). Continuous tracking of intracellular particles in living HEK293 cells was achieved for up to 12 h. Trajectory analysis revealed temporal transitions between directional and diffusive transport, as well as the accumulation of multiple particles within localized intracellular domains over several-hour timescales. The high photostability of Au42, combined with low phototoxicity of NIR excitation, enables visualization of intracellular transport dynamics over timescales difficult to access using conventional visible fluorescent probes. These results establish Au42-based NIR imaging as a platform for long-term, low-phototoxicity intracellular SPT and provide a framework for investigating slow intracellular dynamics in living systems.

There is a vast literature on the voltage gating of ion channels, with a fairly large fraction concerned with potassium channels, especially of the KV1 family, including Shaker. Experimental evidence derived from protein structure has been interpreted to give gating mechanisms that largely disregard water. We propose that the K+ ion, in order to pass through the gating region and enter the cavity pore, must be largely dehydrated. Competitive interactions of each single hydration shell water at the gate, with counterions, protein, or other water molecules, can remove one water at a time. There are several such interactions for the ion hydration shell; for the ion to pass through the gating region, there must be enough such interactions to leave the ion with at most two hydrating water molecules, in which case the gate is open. Protein conformational changes are secondary, small, and mostly unimportant. The hypothesis has a second part: protons, previously shown to be candidate carriers of the gating current (Kariev and Green, JPC B, 2019, Membranes, 2022, 2024) are capable of reaching the gate; adding four protons to the gate prevents dehydration, leaving the ion with at least three hydrating water molecules, enough to block passage. Quantum calculations presented here support the dehydration part of the hypothesis; they also mostly support the second part, concerning the protons, but further work will be required to fully confirm this. The hypothesis explains the experimental finding that the P475D mutant is essentially constitutively open, while the P475S mutant, with a wider gate opening, is closed at all relevant potentials; the computations presented here show the mechanism for this in detail, further confirming the first part of the hypothesis, and largely but not completely confirming the second part, concerning protons, while showing where further work is needed. This mechanism can also qualitatively account for flicker noise and fluctuations, and their consequences.

Light scattering in scintillators is a pervasive problem and a key factor limiting X-ray imaging resolution. Here, we shift scintillator radioluminescence from the traditional visible range into the short-wave infrared (SWIR) or near-infrared II (NIR-II, 1000-3000 nm) window to mitigate light scattering and thereby enhance light penetration and X-ray imaging resolution. We present an NIR II MgGa2O4:Ni2+ scintillator with peak emission at 1340 nm, achieving a threefold improvement in X-ray imaging resolution compared with visible scintillators owing to reduced light scattering. This heavy-metal-free NIR-II scintillator exhibits intense radioluminescence comparable to that of conventional visible-emitting CsI:Tl, achieving a detection limit of 56 nanograys per second, ~100-fold lower than typical doses used in medical imaging. We show that this NIR-II scintillator enables high-resolution X-ray radiography of electronic circuit boards and biological tissues.

Single-molecule localization microscopy (SMLM) enables nanoscale imaging but remains limited in three-dimensional (3D), high-speed, and high-density applications due to background fluorescence, photon inefficiency, and large point-spread function (PSF) footprints. Here, we present single-objective light-sheet microscopy with PSF-engineering enhanced biplane detection (SoLiD-3D), a versatile imaging platform that integrates dual-wavelength light-sheet illumination with dual-color, multi-configuration biplane imaging for parallel acquisition with PSF engineered detection for high-speed volumetric SMLM. Parallelized single-objective light-sheet excitation combined with PSF engineering overcomes key limitations of conventional wide-field and biplane approaches. Independent control of two excitation wavelengths for optical sectioning enables simultaneous dual-target imaging and single-target dual-color imaging with improved contrast and temporal resolution utilizing dynamically displaced light sheets for volumetric coverage. Using SoLiD-3D, we demonstrate high-speed single- and dual-target dual-color imaging that doubles localization density without sacrificing photon efficiency and continuous volumetric imaging via PSF-engineering enhanced biplane detection for whole-cell 3D imaging with improved axial localization performance over extended depth ranges. We further demonstrate improved speed by utilizing the Hummus PSF, a compact engineered PSF that enables high-precision 3D localization with a substantially reduced spatial footprint, for the first time for super-resolution imaging applications. Taken together, SoLiD-3D mitigates the trade-off between axial range and localization precision and offers improved speed compared to conventional 3D SMLM approaches.

Solid-state nanopores offer label-free, real-time single-molecule sensing. However, resolving fast biomolecular transport requires high-bandwidth data acquisition while the intrinsic high-frequency noise limits recovery of informative events. Here we present a hardware-software co-designed nanopore sensing platform that combines wafer-scale low-noise device engineering with deep learning-based signal reconstruction. A low-dielectric SU8 coating on silicon nitride nanopores reduces device capacitance to the pF range and suppresses high-frequency noise by up to 5-fold while maintaining facile, controllable and reproducible fabrication. This extends usable acquisition to 40 MHz and enables capture of fast molecular features. Coupled with a reconstruction model trained on synthetic translocation events embedded in experimentally measured noise, the platform recovers transient sublevels while preserving blockage edges and temporal fidelity. Using engineered DNA molecules carrying dumbbell-like barcodes, we resolve nanometer-scale structural spacings on sub-microsecond timescales, and experimentally quantify translocation dynamics within the sub-10 nanometer regime. Dual-channel measurement on a single nanopore device further demonstrates transferability of the platform by showing robust cross-channel signal reconstruction across distinct baseline noise levels. Our approach provides a general route for reliable recovery of fast event features and may enable more information-rich single-molecule sensing across diverse biomolecular targets.

Direct validation of the nanoscale structural organization of membrane proteins requires localization precision that matches their molecular dimensions. The sodium-potassium pump, or the Na,K-ATPase is an integral membrane protein responsible for maintaining electrochemical gradients and cellular energy homeostasis. Although its crystal structure is characterized, the organization of the Na,K-ATPase within native plasma membranes, particularly whether it forms functional oligomers, remains an open question. Here, we combined 3D MINFLUX nanoscopy with DNA-PAINT with sub-10 nm localization precision to map the clustering topology of the Na,K-ATPase in mammalian cells. By targeting EGFP-tagged Na,K-ATPase 1 and {beta}1 subunits using anti-GFP nanobodies, we obtained high-density 3D localization maps of the protein in the plasma membrane. To evaluate the point patterns, we developed a computational data-driven spatial point assignment approach that segments apical and basal localizations, mitigating clustering artifacts produced by imaging two membranes in close proximity. Furthermore, we used a spatial statistical approach analyzing sequential nearest-neighbour distances to elucidate supramolecular arrangement information. Our data reveal a preferential nearest-neighbour distance of approximately 7 nm, providing direct visual confirmation of Na,K-ATPase dimerization. Additionally, we identified higher-order nanoclusters composed of up to 21 proteins. These findings provide definitive structural evidence of the dimeric configuration of Na,K-ATPase, establishing a foundation for future research on the functional and regulatory implications of Na,K-ATPase clustering.

Scanning ion conductance microscopy (SICM) provides gentle, non-contact cell surface imaging, but it has not been used to investigate intracellular structures because the plasma membrane restricts nanopipette access. Here, we combined SICM with microsonication-based cell de-roofing to expose intracellular actin stress fibers (SFs) in U2OS cells for nanotopographical and -mechanical characterization. Importantly, the de-roofing conditions preserved actomyosin contractility, allowing analysis of SF structural and biomechanical changes during ATP-induced contraction. Resting SFs displayed an average height of 203{+/-}38 nm and width of 357{+/-}73 nm, and a complex surface architecture characterized by regularly spaced long-range height modulations (~500 nm periodicity; Wq ~25 nm) and smaller irregular corrugations (Ra ~19.2 nm). ATP stimulation reduced SF height and width by ~39% and ~15%, respectively, while largely preserving surface corrugation patterns. During contraction, some SFs separated into two longitudinal strands. High-resolution SICM imaging also revealed filamentous crosslinks mechanically coupling neighboring SFs, and nanomechanical measurements demonstrated local stiffening during contraction. These findings provide new insight into the structural and mechanical regulation of SF contraction and highlight the potential of SICM combined with cell de-roofing as a powerful platform for studying dynamic intracellular processes at nanometer resolution.

Poly-L-Lysine (PLL) mediates the non-specific adhesion of cells and is commonly used in Atomic Force Microscopy (AFM) measurements, to ensure that cells remain attached to the substrate. However, it is acknowledged that adhesion affects the measured mechanical properties, in particular in the case Red Blood Cells (RBCs). This results in a wide range of Youngs modulus E reported in the literature. The present study aims at providing a systematic approach to the impact of non-specific adhesion on the rheology of RBCs. It provides a correlation between the topography profile of adherent RBCs and their rheology, from weak (cPLL = 10-3 mg/mL) to strong-adhesion (cPLL = 100 mg/mL) regimes. Using RICM and AFM, we find that there is a continuum of RBC shapes promoted by adhesion, from concave to dome-shaped, as predicted by the theory of vesicle adhesion. Their elastic properties discriminate them into two populations depending on adhesion strength, where stiffer RBCs (E {gtrsim} 100 Pa) correlate with dome-shaped cells. These findings are supported by rheology measurements of the dynamic complex shear modulus G*(f): while the storage modulus increases with cell-substrate adhesion, reflective of an increased membrane shear modulus, the loss modulus remains unchanged. Finally, further analysis inspired by membrane theory shows that different deformation modes may be triggered during indentation of either weakly or strongly adhering RBCs, illustrating the limits of the Hertz model.

Weak magnetic fields affect many biological processes across the tree of life, though the precise molecular sensors and pathways involved in such magnetoresponses remain mostly uncharacterized. Fluorescence is a useful tool for investigating magnetic field effects in flavoproteins, as their chromophores fluorescence intensity can be shown to depend on the spin states of electronic radical pairs. Here, we describe a four-state ordinary differential equation model to understand what parameter sets result in fluorescence contrast between spin states in photocycles with singlet and triplet radical pairs. We conclude that only certain sets of parameters result in the fluorescence intensity being a good proxy measurement for singlet yield. In particular, we observe that the illumination intensity required to obtain fluorescence contrast depends on the rate of the slow spin-independent radical termination reactions that recover ground-state oxidized fluorophores. Moreover, to observe a magnetic field effect in fluorescence intensity when an external magnetic field modulates the singlet yield, the illumination intensity must be strong enough such that photoexcitation is not the rate-limiting step. This understanding suggests that flavoproteins that do not exhibit magnetic field effects in their fluorescence emission under certain experimental setups may still be sensitive to weak magnetic fields in terms of function, as magnetosensitivity in fluorescence depends strongly on illumination conditions.

Precise, non-invasive manipulation of individual living cells remains a central challenge in biomedical science, with far-reaching implications for single-cell analysis, tissue engineering, and the study of cell-cell interactions. Here, we report the first demonstration of single-cell control using bulk acoustic standing-wave acoustofluidics with closed-loop feedback. We introduce VeLO (Vector-based Local Optimization), a model-free, reinforcement learning-inspired algorithm that enables programmable two-dimensional manipulation of individual cells using a single piezoelectric transducer. Without prior calibration or physical modeling, VeLO learns system dynamics online from acoustically induced cell displacements and automatically adapts to nonlinear, time-varying conditions. We achieve robust control across multiple cell types (DU-145, Jurkat, K-562) and independent manipulation of multiple cells, including controlled cell-cell contact. By combining simplicity of hardware with autonomous, adaptive control, this approach establishes multimodal acoustofluidics as a versatile tool for label-free, high-precision single-cell manipulation.

High-quality cellular imaging, especially in live cells, remains constrained by the trade-off among signal-to-noise ratio, phototoxicity, and instrumentation complexity. Here, we report a scalable plasmonic metasurface that generates a spatially ordered array of fluorescence-enhancing near-field hotspots and enables self-supervised denoised, cellular imaging with improved feature readability on a conventional wide-field microscope. The registered hotspot lattice serves as a physics-derived functional prior that identifies where fluorescence amplification is physically grounded and steers neural-network training accordingly, reducing reliance on paired ground truth, large external pretrained models, or extensive supervised datasets. We demonstrate two labeling-density-dependent operating regimes: dense labeling for cytoskeleton structural imaging and sparse labeling for multiplexed sensing of plasma-membrane-associated dynamics across the hotspot array. Our work unites scalable nanophotonic hardware and self-supervised computational imaging into a practical platform for structural bioimaging and on-chip live-cell biosensing under simple wide-field imaging conditions.

Chemical imaging is a powerful branch of modern microscopy encumbered by a lack of flexible, high-throughput analysis tools. Bespoke analytical pipelines typically perform ratiometric analysis on two layers in a multispectral image to describe the relative composition of molecules in a sample. This strategy has been implemented across fields, spanning histopathology, cell biology, environmental science, and materials science. The commercialization of chemical imaging microscopes has facilitated the collection of large multispectral datasets, necessitating accessible ways to process them. This paper describes Multispectral Analysis Graphical User Interface (msaGUI), a desktop graphical user interface to analyze individual and batch datasets of multispectral images. Data is loaded as CSV, TSV, or TIFFs and processed through a user-defined sequence of modular image operations that can be flexibly combined, e.g. to reduce spectral crosstalk or background noise. After analysis, data is visualized as exportable images, histograms, and statistics. To yield publication-quality figures, outputted images are fully customizable. Written in Python with open-source libraries, the msaGUI program is packaged into an executable for Windows and Mac for a fully no-code application. Other operating systems are supported via the Python source code. In summary, msaGUI provides a rapid and user-friendly solution for analyzing and visualizing multispectral data.

Small molecule inhibitors of cell death have wide-ranging potential applications, both as tool compounds in the laboratory and as clinical modulators of pathologic cell death. Previous screening efforts have identified candidate compounds targeting the pro-apoptotic, pore-forming BCL-2 family proteins BAX and BAK, but the complex interactions of these proteins at the mitochondrial outer membrane (with other proteins and the membrane itself) present challenges for compound screening. Although no inhibitors of BAX or BAK have advanced to clinical testing to date, candidate inhibitors have thus far been identified via screening of membrane-containing systems such as liposomes and isolated mitochondria. To address some of the challenges of chemical screening for apoptosis inhibitors, we conducted a small molecule screen utilizing BH3 profiling, a method that quantifies mitochondrial outer membrane permeabilization (MOMP) upon treatment with pro-apoptotic peptides derived from BCL-2 family proteins. Of over 40,000 compounds screened, we identified a series of compounds that prevent MOMP in response to pro-apoptotic peptides. The most potent of these, CDL36, binds to BAX and prevents MOMP at early timepoints. In longer term viability assays, the cytoprotective effect of CDL36 is most potent against death induced by doxorubicin, a widely used chemotherapeutic agent that causes dose-limiting cardiovascular toxicity. Our results elucidate the mechanism of action of new and existing cell death inhibitors, providing a foundation for further development of these inhibitors and potential insights into the mechanisms mediating doxorubicin toxicity in patients.

Quantitative structural analysis by Photoactivated Localization Microscopy (PALM) on the nanoscale is often restricted to fixed cells because motion during prolonged data acquisition distorts image reconstruction. Here, we develop motion-corrected PALM (mcPALM), a live-cell super-resolution approach combining a conventional fluorescence channel with PALM to correct motion-induced spreading of localizations. We further introduce a photoactivation-based correction to estimate molecule numbers from incomplete trajectories. Using PI3P-marked endosomes in yeast as a dynamic model system, we show that mcPALM recovers a live-cell maturation trajectory linking motion-corrected endosome size and calibrated PI3P content, consistent with fixed-cell benchmarks. Unlike fixed-cell PALM, mcPALM preserves endosome dynamics, revealing stage-dependent directed transport and maturation-associated motility shift. Thus, mcPALM extends PALM from static structural measurements in fixed samples to integrated quantification of nanoscale structure, molecular composition and dynamics in living cells. This framework is broadly applicable to other mobile organelles and biomolecular assemblies, enabling live-cell studies on how molecular organization and dynamics are coupled to biological function.