Optica

High-speed three-dimensional imaging in scattering tissues remains challenging because volumetric microscopy generally requires scanning, whereas snapshot light-field approaches divide limited detector pixels among multiple views. This constraint is particularly severe in the second near-infrared window (NIR-II), where commonly used InGaAs cameras typically have relatively small sensor formats and high detector noise. Here we introduce NIR-II squeezed light-field microscopy (NIR-II SLIM), which optically rotates and compresses multiple perspective views before detection, allowing efficient use of camera pixels while retaining complementary spatial information for three-dimensional reconstruction. NIR-II SLIM acquires volumes at up to 600 volumes s-1 with a reconstructed lateral sampling grid of 512 x 512 pixels. We use the method for label-free four-dimensional imaging of cardiac dynamics in pigmented late-larval zebrafish, resolving chamber deformation and millisecond-scale atrioventricular-valve motion, and for NIR-II fluorescence imaging of vascular and lymphatic transport in mice. NIR-II SLIM provides a detector-efficient approach for high-speed volumetric imaging of rapid biological dynamics in scattering tissues.

Studying the role of individual neurons in behavior and disease requires tools for controlling neural activity with high spatiotemporal resolution. Implantable nanophotonic neural probes are capable of delivering targeted photostimulation to enable genetically distinct neurons to be selectively controlled, yet face barriers to achieving scalable emitter densities and lack sensors for monitoring feedback signals relevant to device operation. To address this, we developed laser-integrated nanophotonic neural probes, which feature hybrid-integrated laser diodes (LD) and thermo-optic photonics switches for scalable emitter addressing and on-chip sensors for monitoring optical power and temperature during photo-stimulation. Devices were fabricated at a commercial silicon photonics foundry on 200-mm diameter silicon-on-insulator (SOI) wafers in an active visible-light platform and were controlled using a custom-developed electronic circuit board. Each device features a hybrid-integrated InGaN LD which couples 450-nm light into a reconfigurable photonic switching tree for delivering spatially resolved photostimulation through 16 emitters along a 3-mm implantable shank. Using the on-chip photodetectors, we demonstrate how devices can enable switching tree calibration as well as output power monitoring during photostimulation. Furthermore, using the on-chip temperature sensors, we show how device temperature perturbations resulting from LD and thermo-optic switch activation can be directly monitored during photostimulation to ensure temperature fluctuations remain below 1 {degrees}C. We validate our design by delivering high spatiotemporal photostimulation during an in vivo optogenetic experiment with simultaneous Neuropixels recording to monitor evoked responses. Overall, these scalable integrated devices offer a pathway for neuroscientists to conduct fiberless optogenetic experiments with greater control and precision.

Three-photon microscopy enables deep-tissue imaging but is highly sensitive to excitation pulse quality, since three-photon excitation efficiency scales with the cube of instantaneous intensity. Group-delay dispersion (GDD) and third-order dispersion (TOD) accumulated through the laser and microscope optics can substantially reduce peak intensity at the focal plane, yet these quantities are rarely measured where imaging occurs. Here, we use third-harmonic generation (THG) interferometric autocorrelation, together with Dispersion Look-Up-Table Estimation (D-LUTE) and a joint two-measurement fitting procedure validated on synthetic data, to estimate baseline GDD and TOD directly at the objective focal plane, requiring only a compact autocorrelator module added to the microscope beam path. Applying this method at 1300 nm and 1600 nm excitation across two microscope systems equipped with different units of the same laser model, we find pulse durations 1.4- to 1.7-fold longer than the transform limit at every condition, driven predominantly by TOD, which varied by roughly 2.7-fold between the two systems. Using the endogenous THG signal from myelinated fibers, we further demonstrate in vivo pulse characterization in the mouse brain, finding no measurable broadening between the tissue surface and a depth of nearly a millimeter. This low-cost, easily implemented approach enables routine, in situ pulse monitoring across multiphoton microscopy platforms.

Spectroscopic single-molecule localization microscopy (sSMLM) enables multiplexed super-resolution imaging by simultaneously acquiring the spatial position and spectral information of individual fluorophores. Dual-wedge prism (DWP)-based implementations provide a compact, alignment-stable approach to spectral dispersion, but trade-offs between localization precision, spectral precision, and experimental complexity remain. We systematically compare five DWP-based sSMLM configurations, including two-dimensional (2D) and three-dimensional (3D) implementations using single DWP (DWP-sSMLM) and symmetrically-dispersed DWP (SDDWP-sSMLM). We evaluate lateral precision, spectral precision, and ease of use. SDDWP configurations acquire spectral images in both channels and utilize both for spatial localization, yielding the highest lateral and spectral precision. However, for applications that do not require axial information, 2D-DWP provides a simple, plug-and-play solution with robust performance. This work offers a guideline for selecting DWP configurations based on experimental needs.

Imaging neural fluorescence dynamics during unconstrained behavior remains a major challenge in neuroscience. Head-mounted miniature one-photon microscopes enable in vivo recordings from freely behaving animals. Still, their power and data requirements typically necessitate tethers or heavy batteries, which constrain experiments and may introduce behavioral artifacts. We present Miniscope Zero, a fully wireless miniature microscope platform combining quasistatic cavity resonance (QSCR) wireless power transfer with a high-bandwidth optical data link. The system delivers >500 mW of receiver power across a 2,500 cm2 behavioral arena and streams imaging data at 8 Mbps, supporting real-time data acquisition. Miniscope Zero provides a typical field of view of 600 m x 600 m and a 2.6-fold higher light-collection efficiency than the UCLA Miniscope v4. We demonstrate wireless CA1 GCaMP6f recordings during open-field navigation, enclosed-maze exploration, simultaneous multi-animal imaging, and extended three-dimensional behavior, enabling long-duration cellular-resolution imaging without tether-induced constraints.

While light-sheet microscopy is emerging as a robust method for volumetric imaging with improved axial resolution, its capability regarding two-dimensional, surface-level mapping is often hindered by limitations in data redundancy and reconstruction efficiency stemming from volumetric registration methods. We demonstrate that a multiview imaging approach in an axially-swept, dithered light-sheet microscope paired with computational image reconstruction of view projections is able to address these trade-offs to enable large-scale mapping of surface structural features, leveraging the advantages of multiview light-sheet in scalable field of view, working distance, and near isotropic resolution across the entire imaging depth. To aid in the acquisition and analysis of two-dimensional surface structures, we present a tailored surface mapping workflow and a Fiji plugin for computational reconstruction, promoting robust and comprehensive visualization of surface features of uncleared volumetric samples. Our strategy, termed projection reconstruction for imaging surface morphology (PRISM), integrates axially swept dithered light-sheet microscopy and post-processing software for multiview imaging. The imaging hardware enables near-isotropic resolution across its entire field of view, while the software implementation leverages rigid and affine transformations to align two-dimensional projections of multiview samples. It is designed to work with the BigStitcher pipeline, leveraging its robust algorithm to provide support for two-dimensional image alignment and stitching. We demonstrate the capability of PRISM in studies of lymphatic network mapping in the epicardial layer of intact mouse hearts, as well as surface profiles of FaDu spheroids labeled with antibody-nanodiamond conjugates. This method allows us to quantify cardiac lymphatic branch numbers, diameters, and lengths of a Prox1-tdTomato mouse cardiac model, as well as cluster number and diameters of epidermal growth factor receptor within a FaDu spheroid labeled with a nanodiamond-antibody conjugate, with a significant reduction of post-processing data size. PRISM leverages multiview image projections to promote studies of cardiac lymphatics in mouse models and surface receptor distributions within spheroid models, enabling efficient surface mapping of large, intact, and uncleared biological samples across a variety of scales.

Image scanning microscopy (ISM) is emerging as the natural evolution of confocal laser-scanning microscopy, combining optical sectioning and a two-fold improvement in lateral resolution with high signal-to-noise ratio. Single-photon avalanche diode (SPAD) array detectors enable an effective implementation of ISM by recording a micro-image at each scan position with single-photon sensitivity, negligible readout noise, and without the fixed frame-readout constraints of conventional cameras. Nevertheless, SPAD array-based ISM remains largely confined to laboratories with expertise in microscope development. Here, we present BrightEyes-Scope, a reference implementation of a SPAD array-based ISM system. We provide step-by-step guidelines for optical design, system construction, alignment, calibration, data acquisition, and image reconstruction. Presented as a complete ISM microscope, BrightEyes-Scope also serves as a reference for upgrading existing laser-scanning microscopes. We anticipate that it will accelerate the adoption of ISM for quantitative super-resolution imaging.

Understanding biological function often requires complementary information on molecular identity, morphology, behavior, and physiology acquired simultaneously from the same specimen. Achieving this capability in miniature microscopes remains challenging because integrating multiple imaging contrasts typically increases optical complexity while compromising field of view (FOV), spatial resolution, or form factor. Here we present dual-mode Bio-CM2, a multimodal computational miniature mesoscope that simultaneously captures fluorescence and label-free reflectance through a shared optical architecture based on distributed computational optics. The system enables frame-interleaved, co-registered dual-mode imaging over a [~]7.5 x 10 mm2 FOV with [~]6 m lateral resolution. We demonstrate the versatility of dual-mode Bio-CM2 across diverse biological systems. In freely moving Caenorhabditis elegans, reflectance captures body posture and locomotor behavior that contextualize fluorescently labeled protein aggregates. In freely swimming larval zebrafish, reflectance captures whole-body morphology and swimming behavior, while fluorescence visualizes cardiac activity. In head-fixed mice, simultaneous fluorescence and reflectance imaging across the bilateral dorsal cortex provides complementary measurements of neuronal calcium activity and intrinsic hemodynamic signals. By integrating molecularly specific fluorescence with complementary reflectance contrast, dual-mode Bio-CM2 extends distributed computational optics into a scalable platform for multimodal miniature imaging.

Intracellular dynamics span a broad range of time scales and biomolecular processes, offering insights into cell health, functional state, phenotype, and response to external perturbations. Several label-free optical imaging approaches have been used to capture intracellular dynamics but are limited by spatiotemporal resolution and biomolecular specificity required to distinguish unique subcellular and metabolic processes. In this work, we demonstrate deep-ultraviolet (UV) microscopy as a powerful, label-free, high-resolution approach for quantifying multiscale intracellular dynamics with biomolecular specificity. By leveraging power spectral analysis and phasor analysis, we capture multiscale intracellular dynamics and analyze their UV wavelength-dependent behavior predicated by the absorption of different endogenous biomolecules. We apply this technique to prostate epithelial cell lines of increasing malignancy and reveal quantitative differences in dynamic intracellular activity that correlate with increased metabolic and organelle activity between phenotypes. Furthermore, we elucidate the molecular identities of structures and activity measured via UV dynamics with broad-band coherent anti-Stokes Raman scattering spectroscopy and fluorescence microscopy. We identify lipid-specific structures, and mitochondrial-specific dynamics, among other biomolecular-specific dynamic behaviors. Together, this study demonstrates deep-UV microscopy as a powerful imaging platform for probing spatial and temporally variant intracellular dynamics with biomolecular specificity, with broad implications for cell phenotyping, tissue pathology, and studying new dynamic subcellular processes.

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.

Quantitative fluorescence microscopy requires photon-efficiency, speed and accurate intensity and lifetime measurements. Time-correlated single-photon counting (TCSPC) simultaneously captures intensity and lifetime, but photon pile-up distorts both signals at high count rates, preventing fast acquisitions. Existing corrections discard photons, distort intensity, or require specialized detectors. Here we introduce PURELIGHT, an integrated hardware and software framework that simultaneously recovers undistorted intensities and lifetimes at count rates far beyond conventional pile-up limits. PURELIGHT works with hybrid photodetectors, silicon photomultipliers and photomultiplier tubes while retaining over three times more photons than alternative approaches. Using two-photon imaging, we showcase PURELIGHT's superior accuracy and spatial contrast, demonstrating video-rate subcellular lifetime imaging in awake mice, a unique lifetime-calibrated ratiometric modality and crosstalk-free temporal multiplexing. By removing the limits that have confined TCSPC to low-signal applications, PURELIGHT promotes the adoption of quantitative, photon-efficient microscopy across the life sciences.

Mechanical stretchers are used to physically expand biological samples for microscopic studies of mechanobiology. Existing stretchers suffer from limited strain capacity and non-quantifiable forces, along with focus drift and feature-tracking limitations during microscopy. We develop an iris-based cell stretcher that applies isotropic equibiaxial force to achieve aerial strain of 1664% that corresponds to 4.2x linear expansion for Tensile Expansion Microscopy (TExM), a super-resolution method which increases sample size above the diffraction limit of light. The TExM stretcher uses 3D-printed, printed circuit board (PCB) and cost-effective parts, is portable and automated. We verify the performance of the TExM cell stretcher using image analysis, achieving ~90% mechanical precision, a maximum of four degrees of arm angular deviation, precise speed control down to 0.01 cm/sec, and an average expansion resolution of 3.98 x 10^-3 x. Integrated strain gauge sensors confirm equal application of force by each arm of the stretcher throughout expansion and finite-element simulation and planar-polariscope photoelastic imaging characterize the uniform substrate stress distribution while indicating high stress at the substrate gripping area. An AutoTracking software in communication with the stretcher hardware and microscope enables continuous autofocus and feature tracking during TExM and fiducial markers verify uniform equibiaxial stretch. The stretcher is demonstrated with fixed NIH 3T3 fibroblasts and live HeLa cells, observing ~4x cellular expansion of fixed cell size and separation of live cell clusters, highlighting the potential of TExM cell stretcher for biological imaging.

The ultrastructure of endothelial cells (ECs) "in situ" is of great interest due to their involvement in many physiological processes. In some organs, these cells form transcellular pores or fenestrae, allowing for the rapid exchange of molecules between blood and interstitium. Despite their importance, no optical images of these dynamic morphological structures have yet been acquired in situ. Major obstacles to their in-situ imaging are the lack of specifical labels for fenestrae and their size well below the optical diffraction limit. Here, we report how we have overcome these challenges and managed to visualize the EC ultrastructure in situ in 25 {micro}m thick liver sections. To enable this, a lipophilic, fluorescent membrane dye was infused into the portal vein of murine livers to stain the sinusoidal ECs before the organ was harvested. Tissue sections were subsequently imaged using a novel, super-resolution optical-sectioning structured illumination microscope (OS-SIM), providing approx. 170 nm spatial resolution with significantly faster image acquisition compared to confocal microscopy.

Light scattering in biological tissue limits deep optical imaging. Acoustic waves can modulate optical properties and enhance photon delivery, but source-positioning requirements can complicate integration with other imaging modalities. Here, we present an open elliptical reflector that focuses laser-induced acoustic waves at a spatially separated target while maintaining open optical access. We characterized the spatiotemporal optical modulation induced by the localized acoustic focus. The system increased the local transmitted-light intensity by a factor of 2.57-fold through a 2-mm-thick 1% intralipid hydrogel and enhanced fluorescence signal under two-photon excitation through a 1.5-mm-thick scattering sample. These results demonstrate spatially separated, reflector-based acoustically assisted light guiding for enhanced light delivery and fluorescence excitation through scattering media.

Super-resolution fluorescence microscopy overcomes the optical diffraction limit and has significantly advanced our understanding of biological complexity within the framework of fluorescence labelling (1). Fluorescence labelling underpins this capability, enabling high photon budgets, superior signal contrast, and tuneable photophysical properties essential for diverse super-resolution modalities (2,3). In contrast, label-free Raman imaging offers intrinsic chemical specificity (4), supporting applications ranging from biomolecular fingerprinting to cell metabolic mapping (5-8) and histopathological tissue characterization (9,10). Despite its label-free advantage and chemical specificity, Raman imaging remains fundamentally limited in both spatial resolution and imaging contrast due to inherently low signal throughput and weak intrinsic Raman contrast (11-13). Here, we introduce spatial-filtering nanoscopy (SFN), a physics-driven super-resolution strategy that achieves resolution enhancement through targeted signal purification instead of signal amplification, offering a conceptually distinct pathway beyond the diffraction limit. SFN synergistically integrates a sub-millimetre microsphere lens (SMML) with a standard confocal Raman microscope, harnessing two complementary physical effects: (i) the photonic redistribution effect (PRE), which narrows the lateral excitation profile, and (ii) the three-dimensional spatial filtering (3D-SFE), which effectively suppresses both lateral and axial background. We demonstrate SFN-enabled super-resolution Raman imaging of silicon nanostructures, intact cells, and tissue sections, achieving an effective lateral resolution of approximately 40 nm, and chemically resolving subcellular features including organelles and pseudopodia without exogenous labels. These findings establish SFN as a broadly generalizable hardware-based super-resolution strategy, readily deployable on standard confocal platforms and extensible across diverse optical imaging modalities.

High-resolution, three-dimensional imaging of live organisms has largely depended on laboratory-bound microscopes, limiting quantitative analysis of morphology and dynamics to species that survive transport and thrive under lab-controlled conditions. Here we present a mobile optical coherence microscopy (OCM) platform that overcomes this constraint, delivering [~]2.5 {micro}m axial resolution, label-free, volumetric imaging of live aquatic organisms in both laboratory and remote field environments. We demonstrate the platform across a broad range of aquatic organisms from the lab and field, spanning multiple phyla - including cnidarians, poriferans, annelids, arthropods and echinoderms - resolving internal anatomy, tissue boundaries and organismal morphology at micrometer scale without fixation, fluorescent labeling, or specialized sample preparation. High-speed acquisition, with up to 250 kHz A-line rate and 7.7 Hz volume rate, further enabled morphodynamic imaging of live biological processes, including cellular aggregate motility, embryonic cell division, and organ-level peristaltic dynamics in intact, living animals. To demonstrate field deployability, the platform was operated during the EMBL TREC pan-European expedition, enabling on-site, label-free imaging of marine organisms and plankton immediately upon collection. By decoupling high-resolution volumetric imaging from fixed laboratory settings, mobile OCM opens a path toward field-ready quantitative morphological and dynamic phenotyping of aquatic life.

The lifetime of fluorescent molecules provides an orthogonal readout to fluorescence intensity, opening experimental possibilities of measuring changes in local molecular environments, mechanical tension, and metabolism, among other factors. These changes are best studied live and in vivo; however, limitations of slow imaging speeds, high phototoxicity, and increased data size and complexity have significantly impeded progress on this front. Here, we present a complete and transferable pipeline consisting of a light sheet FLIM microscope and an accompanying machine learning model for data processing that renders long-term and/or high-speed volumetric FLIM (vFLIM) tractable in living systems. We benchmark this pipeline across several biological use cases, model systems, lifetime ranges, and spatiotemporal scales, showcasing a suite of possibilities that our workflow enables. This comprehensive pipeline from imaging to analysis is a crucial step forward towards disseminating the power of live vFLIM to the broader bioimaging community.

Three-dimensional imaging of live Caenorhabditis elegans commonly relies on sequential z-stack acquisition, which can be slow and susceptible to movement and photobleaching. Here, we used light-field microscopy to capture dye and delivery-dependent fluorescence patterns across intact adult worms in a single exposure per channel. With a 40x/1.20 NA water-immersion objective, each reconstructed dataset contained 121 axial slices spanning 109 {micro}m at 0.91 {micro}m spacing. In a representative adult, nematode signal extended from 40.88 to 85.4 {micro}m, corresponding to approximately 44.5 {micro}m of captured axial depth without sequential z scanning. Three fluorescence channels were acquired with a summed exposure time of 112 ms across a 685 x 1010 {micro}m field of view. FM1-43 labelled intestinal, cuticular and vesicular structures, FM4-64 highlighted ingested bacteria and intestinal compartments, and Nile Red revealed vesicular and broader whole-organism fluorescence. Intrinsic blue and green autofluorescence signals were also resolved. These observations demonstrate rapid single-shot acquisition of whole-organism three-dimensional fluorescence information and show how delivery route influences the spatial distribution of commonly used fluorescent probes in C. elegans.

The Transport-of-Intensity Equation (TIE) and polarization differential phase contrast (pDPC) exhibit transfer characteristics that limit their use as standalone quantitative phase imaging modalities. TIE provides stable recovery of low spatial frequencies but attenuates fine structure, while pDPC enhances phase gradients but is intrinsically insensitive near zero frequency. We present a hybrid reconstruction framework that combines a single-shot pDPC measurement with an in-focus-defocused TIE image pair and fuses the resulting phase estimates in the Fourier domain using frequency-selective weighting. The illumination geometry is co-designed to enforce angular separation between modalities, enabling high-frequency phase-gradient acquisition in a single exposure while preserving low-frequency sensitivity through low-numerical-aperture symmetric illumination. The proposed method requires three intensity measurements without requiring interferometric stability or sequential asymmetric illumination. Experimental results demonstrate improved phase fidelity across spatial frequencies, reduced halo artifacts, and enhanced recovery of both smooth and fine structural features. This approach provides a compact and robust method that moves towards extended spatial-frequency phase imaging for dynamic and resource-constrained applications.

Expansion microscopy (ExM) physically magnifies preserved biological specimens by using a specimen-permeating hydrogel to pull biomolecules, such as proteins, apart from each other. ExM protocols exhibit a tension between the need to initially fix proteins to each other during sample preservation, to retain proteins and their nanoscale organization, and the need to separate proteins from each other isotropically, to achieve physical magnification. We here propose to address this tension by fixing proteins directly to the hydrogel network itself, which then serves as a common substrate for both initial fixation and final separation. We equip proteins with chemical handles, then form a specimen-permeating hydrogel that binds the handles, in a ~15-minute chemical process conducted entirely at -20oC. This network fixation ExM (nfExM) procedure enables excellent epitope retention and nanoscale organization preservation, in cultured cells. nfExM may help support ultrastructural investigations of protein complexes and interactions.