Optica

Coherent structured illumination microscopy (c-SIM) is a synthetic aperture optical technique for sub-diffraction limit imaging that extends the utility of traditional SIM to non-fluorescent samples. Here, we present a complementary 5-beam implementation of c-SIM that provides enhanced optical sectioning compared to conventional quadrupolar illumination. Since our approach detects intensity images due to coherent light scattering, it avoids the complications associated with detecting complex fields. Through comparative measurements on calibration samples and live microalgae, we show that 5-beam c-SIM effectively suppresses coherent defocus effects, improving image quality while simultaneously providing a 2-fold lateral resolution improvement.

Light-sheet fluorescence microscopy (LSFM) has revolutionized biological imaging by enabling high spatial and temporal resolution with minimal photodamage. However, conventional LSFM techniques often suffer from striping artifacts in the resulting images due to light scattering and absorption within samples, leading to uneven illumination that negatively impacts the accuracy of subsequent image analyses. To address this limitation, we introduce dodecagon light-sheet fluorescence microscopy (dodecaLSFM), a novel approach that maximizes angular diversity to achieve homogeneous illumination and suppress striping artifacts. dodecaLSFM employs diffraction optics and cylindrical lenses to generate twelve light sheets, providing 360 degree omnidirectional illumination that significantly enhances illumination uniformity compared to traditional mSPIM, mDSLM, and ultramicroscopy systems, which use only one or two illumination planes. We demonstrate the effectiveness of dodecaLSFM by achieving high-resolution imaging of whole mouse brain vasculature following tissue clearing, allowing precise morphometric analysis of vascular networks without striping artifacts. Furthermore, we show that combining dodecaLSFM with expansion microscopy (ExM) enables whole-organ 3D imaging at cellular resolution. This novel approach provides an advanced, scalable solution for large-volume imaging, facilitating detailed structural and functional studies across diverse biological applications.

Minimally invasive surgery is a powerful technique that enables operations deep within the body while minimizing patient trauma and recovery time. Optical endoscopes are key to providing intraoperative vision but still face challenges due to the loss of essential senses, including depth perception and tactile feedback for tissue evaluation. Thus, it is critical to develop endoscopic imaging technologies that can augment operators with critical information. In this work, we explore a prototype multimodal 3D imaging endoscope that integrates volumetric light-field imaging with laser-speckle contrast imaging to simultaneously capture 3D structure and blood-flow information in a clinically relevant form factor.

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.

Cellular metabolic activities can be studied using label-free vibrational spectroscopic imaging to leverage the endogenous contrast of biomolecules. However, fast live-cell imaging over large fields-of-view remains challenging due to the need for raster scanning and despite advances in wide-field modalities, imaging rapid cellular activities across large cell populations remains challenging. Here, we introduce a mid-infrared optothermal microscopy method termed Instantaneous Phase-Shifting Optothermal Microscopy (IPSOM). IPSOM circumvents conventional mechanical phase-shifting methods, achieved single-frame imaging speed 588-fold increase compared to the sequential approach, under field-of-view of 300x350 {micro}m. For multi-wavelength hyperspectral imaging, IPSOM achieves an 8-fold speed improvement. IPSOM is used here to monitor lipid remodelling in adipocytes during lipolysis, demonstrating its potential for studying rapid cellular metabolism.

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.

Optical fluorescence microscopy enables visualization of biological structures and dynamics. However, the intrinsic diffraction limit, especially axially, and depth-related scattering noise compromise the image resolution and fidelity. Computational 3D deconvolution is a promising approach for mitigating these issues, yet its execution is hindered by inaccurate and cumbersome theoretical modeling or experimental measurement of 3D point spread function (PSF), as well as ineffective 3D noise regularization. Furthermore, in the 3D super-resolution regime, there remains a lack of standardized tools for evaluating 3D super-resolution fidelity. Here, we present the 3D adaptive deconvolution and evaluation (3D-ADE) toolkit, which comprises 3D-Ada deconvolution with physics-oriented automatic 3D-PSF calibration, and 3D-SQUIRREL for 3D super-resolution quality assessment. It effectively resolves noise instability, eliminates the need for 3D-PSF calibration, and reliably assesses the fidelity of 3D resolution extension via deconvolution, physical, and deep-learning-based methods. Accessible via multiple software platforms, 3D-ADE enhances the versatility of 3D deconvolution and fills the gap in 3D super-resolution evaluation tools, and thereby advances volumetric fluorescence imaging applications.

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.

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.

Urinary stone disease is a common urological condition with increasing incidence, particularly in developed countries. Laser lithotripsy (LL) has become a preferred minimally invasive treatment due to its high precision and low tissue damage. Recent studies suggest that cavitation plays a critical role in stone damage during LL, and three-dimensional passive cavitation mapping (3D-PCM) has emerged as a promising tool for detecting these events. However, clinical translation of 3D-PCM remains challenging due to limitations in imaging depth, field of view (FOV), and procedural compatibility. Here, we present a large-FOV dual-modality imaging system (3D-PCM and B-mode ultrasound) based on a large-aperture planar ultrasound array. Through array optimization and model-based reconstruction, our system achieves an expanded FOV of ~40*40mm^2 at a clinically relevant imaging depth of ~110mm, while maintaining high spatial resolution of ~0.6 mm laterally and ~0.4 mm axially. In vivo experiments in a porcine model demonstrate that the reconstructed cavitation distribution correlates well with stone damage. Our technology has the potential to provide real-time treatment feedback during LL without disrupting the standard workflow.

Tissue-clearing and light-sheet microscopy have transformed volumetric imaging of intact organs, yet limited mechanistic understanding of dehydration-based clearing continues to constrain rational protocol design and broader applicability. Here, we define the cardinal chemical and physical principles underlying dehydration-based tissue-clearing and establish a new pipeline for large-volume imaging. To maximize imaging performance, we developed the mesoSPIM-ultra, an upgraded mesoSPIM platform with a temperature-controlled sample chamber, a large field-of-view (FoV) camera and specialized optics to achieve long-working-distance, high-resolution imaging of cleared samples. We applied this approach to investigate the projectome of Chx10+ neurons, a cell population with complex axonal morphologies along the entire mouse spinal-cord and brain, and implicated in ipsilateral orienting behaviors. By combining behavioral analysis with post-hoc single-neuron reconstructions, we revealed previously inaccessible branching architectures and long-range projections extending from the brainstem to the spinal cord. Together, our work establishes a mechanistic foundation for tissue-clearing and scalable 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 lifetime imaging microscopy (FLIM) provides molecular contrast that is largely independent of fluorophore concentration, yet it remains constrained by a persistent trade-off among acquisition speed, photon dose, and detector complexity. To address this challenge, we developed image-projection fluorescence lifetime imaging microscopy (IP-FLIM), an integrated optical and computational platform that enables high-resolution, component-resolved lifetime imaging using only a linear single-photon avalanche diode array. We validate IP-FLIM using fluorescent microbeads and bovine pulmonary artery endothelial cells, demonstrating up to 22.3x improvement in contrast-to-noise ratio and 72.3% reduction in background noise over conventional filtered back-projection reconstruction. By combining wide-field projection acquisition with computational k-space reconstruction, IP-FLIM provides a scalable route to fast, high-resolution multiplex lifetime imaging.

Significance: Continuous, noninvasive, and depth-resolved monitoring of blood-flow-related tissue dynamics remains an important unmet need. Speckle-contrast optical spectroscopy (SCOS), including interferometric implementations such as iSCOS, provides a scalable optical route to blood-flow sensing, but conventional continuous-wave approaches lack intrinsic depth selectivity. Time-of-flight (TOF) gating offers a way to separate superficial and deeper dynamic contributions in layered tissues, such as skin-muscle or scalp-cortex, by resolving photon path lengths. Aim: We introduce a swept-source, single-channel implementation of interferometric speckle-contrast optical spectroscopy (iSCOS) to obtain TOF-resolved temporal speckle contrast, {kappa}^2, from the measured field autocorrelation g_1, and evaluate its feasibility for depth-resolved blood-flow sensing. Approach: A swept-source iNIRS system operating at 780 nm acquired interferometric signals, which were Fourier-transformed along the optical-frequency axis to recover complex TOF-resolved speckle fields. Temporal speckle contrast was then estimated at each TOF gate indirectly from g_1 using the speckle-visibility relation. Diffusion-based numerical simulations were first used to compare the direct variance-based estimator and the indirect g_1-based estimator under varying reduced scattering coefficient, diffusion coefficient, additive noise level, and bi-layer geometry. Because the simulations showed that the g_1-derived {kappa}^2 estimator was substantially less sensitive to additive noise than the direct estimator, this estimator was used for the main phantom and in vivo analyses, while the direct estimator served as a simulation comparator. The g_1-derived estimator was then applied to liquid and bi-layer phantoms, followed by proof-of-concept in vivo measurements on the human forearm during cuff occlusion and on the forehead during a Sudoku task. Results: TOF-resolved kappa2 curves recovered with the g_1-derived estimator matched DWS theory across scattering coefficients, photon path lengths, and exposure times. The estimator preserved theoretical accuracy for additive noise amplitudes up to 50% of the field amplitude, whereas the direct variance estimator showed substantial noise-induced bias and required correction. Bi-layer simulations and phantom experiments reproduced the predicted direction and onset of TOF-dependent decorrelation-rate trends in layered media. In vivo, the recovered blood-flow index tracked the expected TOF-dependent cuff-occlusion and reactive-hyperemia response in the forearm. During the single-subject Sudoku task, the left-forehead recording showed a TOF-dependent relative blood-flow-index increase of +0.8 {+/-} 1.9% at TOF = 400 ps, +9.8 {+/-} 2.2% at TOF = 600 ps, and +15.2 {+/-} 5.6% at TOF = 800 ps. This pattern is consistent with increased sensitivity to deeper tissue at longer photon path lengths, but requires cohort-level validation before quantitative interpretation as cognitive activation. Conclusions: Coupling temporal speckle-contrast analysis with swept-source iNIRS yields a proof-of-concept, depth-resolved platform for blood-flow sensing. By estimating TOF-resolved speckle contrast through the g_1-derived {kappa}^2 route, TOF-iSCOS suppresses additive-noise bias while preserving sensitivity to deeper dynamic tissue layers. The present single-channel results bridge continuous-wave iSCOS, interferometric NIRS and time-domain diffuse correlation spectroscopy (TD-DCS), and motivate future multi-channel and cohort studies for scalable cortical hemodynamic monitoring.

In vivo infrared thermography is limited by the inherently poor spatial resolution at long wavelengths, low contrast, and the lack of biocompatible contrast agents. Here, we present 3-5 m mid-wave infrared (MWIR) thermography enhanced by an artificial intelligence (AI) network and cold phosphate-buffered saline (PBS) as a thermal contrast agent for noninvasive in vivo imaging with high contrast and resolution. MWIR imaging enabled high thermal sensitivity with microscale spatial resolution, strong relative thermal contrast, and facilitated visualization of the subcutaneous vasculature in the human arm, hand, ankle, the femoral artery and vein in rats, and the femoral vessels in mice, with image contrast further enhanced by AI networks. In a 4T1 tumor-bearing mouse model, AI-enhanced MWIR resolved early-stage tumors of ~2.3 mm and metastases as small as ~1.7 mm. Using cold PBS as a MWIR thermal contrast agent, we achieved precise tumor boundary visualization and real-time imaging-guided tumor resection. AI-enhanced MWIR offers a promising solution for early diagnosis and improved surgical precision.

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.

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.

This study reports a brand-new continuous-wave-excited (CW-excited) two-photon fluorescence emission mechanism in indocyanine green (ICG), an organic fluorescent dye widely used in clinical practice. This mechanism is based on the excited state absorption (ESA) process of the first singlet excited state. Intramolecular electrons sequentially absorb two photons to reach a high-energy singlet excited state, followed by direct radiative transition to the ground state to generate fluorescence. The entire process is exclusively mediated by singlet states. We further summarize the essential requirements for organic dyes to realize this luminescence mechanism. First, the dye must possess at least two well-separated singlet excited states with distinct energies, corresponding to two absorption peaks at different wavelengths in the absorption spectrum. The wavelength of the high-energy singlet excited state is about half that of the first singlet excited state. Second, the peak in the absorption spectrum corresponding to the transition from the ground state to the first singlet excited state has a sufficiently large molar extinction coefficient. Third, the first singlet excited state exhibits the capability of ESA. Fourth, electrons at the high-energy singlet excited state can directly transit to the ground state and emit fluorescence. We validated this mechanism in a variety of organic dyes satisfying the above conditions, confirming its universality. Using CW laser as the excitation source, we achieved two-photon fluorescence imaging of mouse cerebral blood vessels at a depth of 400 m, which clearly resolves three-dimensional vascular networks with high resolution. We also performed two-photon fluorescence imaging on human gastric cancer tissue samples at a depth of around 150 m, which provides a low-cost strategy for clinicians to rapidly acquire high-contrast tumor tissue images.