ACS Photonics

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.

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.

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.

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-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.

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.

Point-spread-function engineering by depth-encoding phase masks enables volumetric super-resolution imaging by 3D single-molecule localization microscopy (SMLM) but usually requires cumbersome relay optics. We demonstrate simple 3D SMLM implementation by phase mask insertion directly into the infinity space of a commercial microscope, along with appropriate computational compensation for field-dependence. The entire inserted component, including the phase mask, is 3D printing-based.

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.

Visual neuroscience requires precise spatiotemporal projection of optical stimulation onto the retina, especially in experimental mouse models. However, in vivo patterned stimulation in mice is profoundly hindered by the extreme optical power and severe anatomical aberrations of the eye. Consequently, visual stimulation relies mainly on unverifiable, open-loop approximations that often lack spatial precision. Here, we introduce a closed-loop, spatially modulated stimulation platform that overcomes these barriers. By integrating a digital micromirror device (DMD) with electronically tunable lenses (ETLs) and a real-time, fundus camera-guided focus optimization module, we directly verify the location of patterned stimuli on the retina while dynamically correcting for chromatic and geometric defocus. This platform delivers quantitatively verified static and dynamic patterned stimuli to the living retina with lateral resolutions as fine as 6.7 {micro}m. Guided by ray-tracing optical analysis, our work establishes a technological foundation that enables highly reproducible, cellular-scale interrogations of the visual pathway.

Deep learning has enabled single-molecule localization microscopy (SMLM) at high emitter densities, but only for single channel systems. Here we present DECODE-Plex, a deep-learning-based framework to localize dense single molecules with overlapping point spread functions simultaneously in multiple channels. We showcase DECODE-Plex on experimental ultra-high density dual-color and 3D live-cell data. Packaged for ease of use, it will enable many groups to improve imaging speed and quality of multi-channel SMLM.

Surface phenotyping underpins plant science, preclinical animal research and entomology, yet across all three the measurement is almost always a photograph, which records a projection and not the surface itself. Here we present the Gentschinator3000, an open structured-light platform that brings high-end metric surface measurement within reach of laboratories with no optics expertise, combining documented open hardware, open reconstruction software and analysis workflows for under 4000 Euro in components. It resolves a planar reference to 45 m local flatness, registers full rotations to a loop closure of 156 m, and performs stably across acquisition ranges that we define. Applying one workflow to a leaf before and after desiccation, to murine anatomy and to a spread lepidopteran, we find that projection underestimates surface area by 11 to 41 %. That error grows with the condition under study, with the evaluation scale and with the direction of view, so it can confound phenotype comparisons dramatically. In murine limbs a 15-degree change of viewing direction shifts a projected inter-segment angle by up to 23.2 degrees, while the three-dimensional angle does not move. Projection geometry can therefore contribute as much to a measured phenotype as the biology it is meant to quantify.

Microlasers based on whispering-gallery modes (WGMs) provide bright, spectrally narrow emission, making them ideal optical probes for high-resolution cellular tagging, single-cell tracking, and biosensing. To reproducibly apply WGM microlasers in biomedical or tissue-engineering contexts, control over the number of lasers in a cell population is highly desirable. Here we use a commercial instrument for flow cytometry-based cell sorting to obtain HEK293T cell populations with a controlled number of polystyrene laser particles (LPs). By modifying the particle surface with poly(ethyleneimine) (PEI) or lipofectamine, we enhance LP-association to cells. We demonstrate that our sorting procedure yields viable cells showing unchanged proliferation rates. Manufacturer-dependent differences in particle-cell interactions were observed and investigated by scanning and transmission electron microscopy (SEM/TEM) and flow cytometry. Finally, we show that the same sorting approach enables the generation of multi-cellular spheroids with a defined ratio of LP-containing and LP-free cells. This work lays the foundation for larger tissue engineering studies with defined LP populations and control over LP-placement with respect to the cell.

Joint optical measurement of hemoglobin and blood-flow dynamics could provide a compact route to richer functional monitoring of tissue physiology. However, hybrid diffuse-optical systems commonly optimize photon time-of-flight (TOF) for individual readouts, even when absorption and flow are recovered from the same acquisition. Here, we asked whether a single TOF window can optimally preserve both signals. Dual-wavelength TOF-iSCOS recovered absorption, hemoglobin and relative blood-flow index (BFI) from the same interferometrically sensed optical field, while Early, Middle and Late photon windows provided progressively greater weighting of longer and deeper photon paths. During forearm cuff occlusion in 11 adults, absorption and flow showed markedly different TOF dependence: Late-gate BFI responses were retained, whereas absorption decreased to a median Late/Early ratio of 0.18. The resulting readout-by-TOF interaction was positive in all 11 participants (median 2.61 octaves, 95% bootstrap CI 2.44-3.22; exact sign-test P = 9.8 x 10-4). Readout-specific gate selection increased held-out response retention by 24.8% (95% CI 8.8-42.5%) relative to a single common gate. We then tested the approach during prefrontal working-memory activation in seven participants using a higher-load 2-back task relative to a low-load 0-back control. Late-gate BFI increased in all seven participants (+5.07%, 95% CI 1.46-8.68%; P = 0.014) and remained positive after adjustment using an Early-TOF signal, whereas hemoglobin responses were less consistent. Layered Monte Carlo modeling showed that the same detected photon histories acquire different absorption and dynamic-scattering sensitivity across TOF, linking the experimental effects to anatomy- and geometry-conditioned tissue weighting. Within the present geometry and photon budget, the central result is therefore not that one readout is intrinsically superior, but that absorption and flow recovered from the same optical field retain physiological information differently across photon time-of-flight.

Significance: Fluorescence imaging remains largely qualitative and device-specific, limiting reproducibility and intersystem comparisons. SI-traceable radiometric characterization requires calibrated sources suitable for transferring known radiance to imaging systems. Aim: Establish and evaluate a compact, calibrated solid-state radiometric emitter target (RET) for SI-traceable radiance transfer, and assess methods for evaluating radiance invariance, imager responsivity, and extension of radiance transfer to digitally defined regions of interest (ROIs). Approach: The radiance invariance of an LED-based RET was evaluated by measuring its radiance across varying source-to-aperture distances and collection apertures. A previously described radiance-transfer formalism based on calibrated source radiance and collection geometry was implemented to determine imager responsivity (R{lambda}), which was evaluated across varying working distances and lens apertures. Grubbs and MAD outlier tests and one-way ANOVA were used to assess radiance and responsivity invariance. Finally, responsivity obtained using digitally defined ROIs was compared with matched physical apertures to assess ROI-based radiance transfer. Results: The measured RET radiance remained invariant across the tested source-to-aperture distances and collection apertures, with no significant differences observed across distance (p = 0.88) or aperture (p = 0.15). Imager responsivity remained stable across working distances for both tested lens configurations (p = 0.48 and p > 0.99). Responsivity was also invariant across lens apertures, except for deviations at f/11 that were consistent with practical aperture tolerances. Digitally defined ROIs reproduced responsivity values obtained using matched physical apertures, demonstrating that the radiance-transfer approach can be extended to image-defined regions. Conclusions: The presented methods provide an approach for evaluating compact LED-based sources for invariant-radiance emission and suitability for SI-traceable radiance transfer. The characterized RET supported stable imager responsivity across changes in imaging geometry and extension to digitally defined ROIs. These methods can be adapted to other LED-based sources, with further angular characterization and uncertainty analysis needed to establish broader applicability.

Multispectral imaging is a cornerstone of chemical imaging, with bioluminescence, fluorescence, and phosphorescence imaging underpin much of preclinical and analytical chemical measurements. However, conventional implementations are typically proprietary, costly, and resolve spectral content from the reflectance of a broadband source, without control over the excitation spectrum. This architecture cannot isolate excitation-dependent photophysical processes. Spectral discrimination through sequentially resolved narrowband excitation, detected on a single broadband-sensitive camera, instead enables excitation-dependent fluorescence, phosphorescence, and reflectance measurements not available to such broadband approaches. We describe AURORA-MSI, an open-source multispectral platform that inverts this arrangement with fourteen narrowband LEDs spanning 367-940 nm, a 120-element annular illumination ring with eight independently addressable azimuthal sectors, and a thermoelectrically cooled monochrome CMOS camera. Radiometric calibration, spatial uniformity mapping, and camera noise characterization establish the quantitative measurement foundation. In bioluminescence imaging, AURORA-MSI localized sources in a calibrated tissue-mimicking mouse phantom comparably to a commercial Revvity IVIS Spectrum, detected luciferase-expressing HSJD-GBM1-001 glioblastoma cells across a dilution series, with reduced replicate consistency at the lowest densities, and mapped substrate-free fungal-pathway emission in an intact bioluminescent Petunia hybrida, none requiring photon-counting instrumentation. Time-resolved phosphorescence decay imaging of six inorganic phosphors over 33 minutes found tri-exponential models adequate at early times, while distributed-lifetime models were preferred at extended timescales. Multispectral image-texture features extracted across all fourteen excitation bands discriminated ten pharmaceutical and household powders spanning distinct chemical compositions and, in two cases, distinct formulations of the same compound. Generality beyond these regimes was established through dual-excitation fluorescence fingerprinting of fifteen mineral specimens and wavelength-selective plant-tissue imaging exploiting UV and NIR penetration-depth differences. Excitation-side spectral encoding enables photophysical measurements that detection-side systems with uncontrolled broadband illumination cannot isolate, and the cooled-camera architecture supports weak-signal modalities without photon-counting instrumentation.

Fluorescence lifetime imaging microscopy provides quantitative, concentration-independent contrast for probing molecular interactions, biochemical environments and cellular physiology. However, the requirement to acquire sufficient time-resolved photon statistics makes FLIM inherently slow, limiting its application to dynamic biological processes. In live-cell applications, including calcium signalling and vesicular trafficking, acquisition times can exceed the timescale of the underlying biology, causing temporal averaging, motion artefacts and loss of transient information. Methods that increase FLIM acquisition speed while preserving quantitative lifetime accuracy are therefore required. We report on a high-speed, compressive, multiphoton fluorescence lifetime imaging technique (TomoFLIM). Two-photon fluorescence is excited using a line focus projected tomographically across the sample, while time-tagged fluorescence is acquired using time-correlated single-photon counting. Time-resolved fluorescence data are reconstructed using Lucy-Richardson deconvolution followed by a computationally efficient centre-of-mass method lifetime estimator. In addition, TomoFLIM Net, a physics-informed neural-network, directly reconstructs fluorescence intensity and lifetime from compressed time-resolved tomographic data. TomoFLIM was benchmarked against raster-scanned fluorescence lifetime measurements using calibrated fluorescence lifetime beads and biological specimens. We demonstrate imaging at compression ratios exceeding 90%, with Pearson correlation coefficients above 80% relative to reference images. A raster-scanned FLIM dataset acquired in 60 s was reproduced using TomoFLIM in 3.75 s, representing a 16-fold increase in frame rate and equivalent reduction in accumulated dark counts. TomoFLIM Net recovered distinct experimental bead lifetime populations, demonstrating a direct route from compressed measurements to quantitative lifetime maps. TomoFLIM therefore offers significant potential for rapid live-cell imaging of dynamic biological processes, including deep within turbid biological specimens.

The sea anemone Aiptasia is a model system for understanding cnidarian loss of symbiotic algae under heat stress (bleaching). While Aiptasia polyps have been widely used to study this process, accurate symbiosis phenotyping grapples with discordant length scales: fine spatial resolution (~100 um) is needed across a whole organism (~5 mm). To address this, we consider small (~100 um), optically transparent Aiptasia larvae as a bleaching model suitable for whole-organism phenotyping by fluorescence microscopy with larvae classified as symbiotic when algae are localized within gastrodermal cells. To expedite phenotyping, we introduce a machine-learning (ML) image-analysis pipeline (SYMPHONY) designed for single-larva resolution analysis of intact larvae. SYMPHONY efficiently identifies the cellular location of internalized algae (accuracy: 79%, precision: 82%, recall: 79%, F1 score: 79%; training dataset composed of 1611 total objects). Additionally, SYMPHONY reports statistically significant larval bleaching under heat stress and corroborates manual phenotyping results, while significantly reducing operator labor from hours to minutes. The combination of the Aiptasia larvae model and the SYMPHONY pipeline aims to accelerate our understanding of symbiosis breakdown.

Compact fluorescent proteins (FPs) with red-shifted emission are needed for deep-tissue short-wavelength infrared (SWIR) imaging. We engineered a GAF domain from the JSC1 cyanobacteriochrome of thermophilic Leptolyngbya sp. into three monomeric, biliverdin-binding FPs of 19.1 kDa: miRFP729nano, miRFP732nano and miRFP735nano, with excitation/emission maxima of 714/729, 716/732 and 719/735 nm, respectively. Their off-peak fluorescence beyond 1,000 nm was several-fold higher than that of miRFP718nano previously used for SWIR imaging. miRFP732nano functioned as a fusion tag, a component of target-stabilized nanobodies, and a reporter of NF-{kappa}B and AP-1 transcriptional activities. It enabled single-laser, dual-color three-photon imaging with EGFP to depths of [~]950 m in cortex and [~]300 m in spinal cord. In mice, miRFP732nano supported SWIR imaging of skeletal muscle, inflammatory signaling and intracellular targets. Combining SWIR detection with biocompatible 4-aminoantipyrine-based in vivo tissue clearing enhanced signal and image sharpness. These red-shifted NIR FPs expand the genetically encoded toolkit for deep-tissue imaging.

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.

Fluorescence imaging in the second near-infrared window (NIR-II, 950-1700 nm) offers reduced scattering, lower autofluorescence, and deeper tissue penetration than NIR-I imaging, but its adoption is limited by the need for custom-synthesized fluorophores. Here, we identify commercially available dyes that exhibit usable NIR-II emission. Eleven visible, far-red, and NIR-I fluorophores were screened under twelve acquisition configurations combining 670, 760, and 808 nm excitation with band-pass (950 nm, 1400 nm) or long-pass (1000 nm, 1250 nm) emission filters. Output varied markedly with fluorophore identity and excitation/emission configuration. Among hydrophobic dyes, DiR exhibited strong emission across almost all excitation and emission filters. Among hydrophilic dyes, strong NIR-II fluorescence was observed for IRDye 680RD (excitation at 670 nm), sulfo-cyanine 7 (excitation at 670 nm and 760 nm), and indocyanine green (excitation at 808 nm). DiR showed a linear concentration-response under 760 nm excitation with BP1400 detection. Upon encapsulation in PEGylated liposomes, strong NIR-II fluorescence was retained. In an in vivo study in mice, NIR-II resolved vasculature that NIR-I could not consistently delineate, and enabled pharmacokinetic analysis. Both windows returned similar ex vivo organ distributions. NIR-II imaging is therefore accessible using commercial off-the-shelf fluorophores, provided the dye is matched to the intended excitation/emission configuration.