Optica

Long-Stokes-shift fluorophores enable high sensitivity and multiplexed imaging with single-wavelength excitation. Under single-photon illumination ATTO 490LS exhibits a 165-nm Stokes shift, but its two-photon properties remain uncharacterised. Emission and excitation spectral analyses of ATTO 490LS in ex vivo Drosophila melanogaster brains identified two-photon excitation sensitivity at 940 nm, with peak emission at 640 nm. We demonstrate successful duplexed imaging of ATTO 490LS alongside Alexa Fluor 488 using a single 920-nm fibre laser and dual photomultiplier tubes, enabling distinct measurement of red and green fluorescence signals. These findings establish ATTO 490LS as suitable for multicolour two-photon microscopy with single-laser systems.

Obtaining structural information from the enteric nervous system (ENS) within intact intestinal tissue requires microscopy systems capable of imaging through multiple tissue layers and during ongoing physiological motion. Tissue opacity, three-dimensional geometry, and spontaneous contractions strongly constrain volumetric imaging, limiting the applicability of most conventional linear optical techniques to imaging in either dissected, stretched or pharmacologically suppressed tissues. We apply Spectro-temporal Laser Imaging by Diffracted Excitation (SLIDE) microscopy, a diffraction-based scanning approach enabling fast volumetric two-photon imaging, to record the ENS in an intact ex vivo intestinal preparation from a transgenic mouse line expressing the red fluorescent protein TdTomato in peripheral and enteric neurons and glia. We achieved fast volumetric imaging during spontaneous contractions, capable of resolving micrometer-scale displacements in three dimensions, without inducing observable photodamage or compromising tissue viability over the experimental timescale. This work establishes 4D-SLIDE microscopy as a robust experimental framework for visualizing enteric neural structures within their native three-dimensional context during physiological motion, with direct relevance for conditions involving altered intestinal mechanics.

Volumetric fluorescence microscopy is a powerful method for studying complex biological systems because it enables comprehensive observation of structural and physiological dynamics. In particular, light-sheet microscopy (LSM) is a leading option for real-time volumetric fluorescence imaging as it combines high imaging speed, low phototoxicity, minimal photobleaching, high spatiotemporal resolution, and low computational burden. To capture fast biological events, various efforts have been made to improve the imaging speed of volumetric fluorescence microscopy, including LSM. However, existing approaches entail significant trade-offs that make routine volumetric imaging at and beyond video rates challenging under practical conditions. Here, we introduce image-scanning LSM, a method that substantially increases the volumetric imaging speed achievable with LSM while preserving key performance metrics, such as spatial resolution and photon efficiency, as well as accessibility. Our implementation, termed image-scanning oblique plane (ISOP) microscopy, enables volumetric fluorescence imaging at up to 1,000 volumes per second with submicrometer lateral spatial resolution. We demonstrate the broad utility of ISOP microscopy by recording and analyzing the dynamics of behaving and rapidly moving organisms.

Optical scattering in biological tissues fundamentally limits fluorescence imaging by disrupting spatial and angular information, thereby restricting volumetric visualization. Although hardware-intensive and computational approaches have advanced scattering microscopy, practical three-dimensional imaging through tissue remains constrained by instrumental complexity and axial ambiguity. Here, we present volumetric scattering microscopy (VSM), a scan-free, optical-computational framework for three-dimensional fluorescence imaging in scattering biological media. VSM captures angularly resolved speckle-encoded fluorescence using an aperture-segmented Fourier light-field configuration and reconstructs volumetric structure through adaptive feature-based descattering and joint sub-pupil alignment. This hybrid strategy preserves angular information embedded in scattered light without wavefront measurement or mechanical scanning, while maintaining the simplicity of a standard epi-fluorescence architecture. We demonstrate high-fidelity volumetric reconstruction across phantoms, engineered cellular systems, ex vivo tissues with volumetric muscle loss, and intact Xenopus embryos, achieving preserved spatial resolution, enhanced optical sectioning, and quantitative accuracy under strong scattering conditions. VSM supports large-field, robust volumetric imaging in both layered and fully embedded scattering environments. By transforming scattered light into a structured encoding resource, VSM establishes a scalable pathway toward routine three-dimensional fluorescence imaging in complex biological systems.

The performance of a laser scanning microscope inevitably depends on the performance of the point detector. As laser scanning approaches aim to penetrate deeper in tissue, there is a commensurate need for detectors that can operate with high sensitivity, bandwidth, and dynamic range at near-infrared wavelengths where scattering is reduced. Here, we demonstrate that fiber optical parametric amplification can be used to boost low-power microscopy signals to levels that can be detected by near-infrared photodiodes without introducing prohibitive noise. We construct amplifiers that achieve >50 dB of parametric gain at wavelengths within the third near-infrared transparency window and have similar sensitivity to near-infrared photomultiplier tubes. Furthermore, these amplifiers outperform detection with a photodiode and subsequent electrical amplification, providing a factor of 10-100-fold improvement in sensitivity. We demonstrate amplifier bandwidths up to ~1.6 GHz, a factor of 10 faster than conventional detectors, including near-infrared photo-multiplier tubes, with sensitivity of ~8 nW (corresponding to ~20 photons/pixel). Finally, the increased performance of the optical amplifier is confirmed in diagnostic imaging experiments where >10x less power is required to achieve the same signal-to-noise ratio and contrast as images using electrical amplification. Accordingly, fiber optical parametric amplification is a new path forward for extending the performance of laser scanning microscopes in the near infrared.

The eye offers a unique non-invasive window for accessing single-cell level structures and functions of the central nervous system (CNS) throughout the retina. However, strong and space-varying ocular aberrations, along with limited volume rates, challenge large-scale cellular imaging in living eyes and stymie the full potential of possible biological and pathological studies in retina. Here, we present plenoptic illumination scanning laser ophthalmoscopy (PI-SLO), a 3D fluorescent retinal imaging modality that enables high-speed, widefield, volumetric single-cell imaging with low phototoxicity. By capturing multiple angular images of fluorescence signals from the entire volume, PI-SLO enables digital aberration correction and 3D imaging across a >20{o} FOV with >23 Hz volume rate. We leverage this structural and functional imaging modality to investigate three key aspects of CNS physiology through the living mouse retina, including: microglial process dynamics, vascular perfusion, and light evoked calcium fluxes in inner retinal neurons. PI-SLO is a versatile non-invasive platform for in vivo investigation of retinal and CNS physiology at the cellular level.

Fourier ptychographic microscopy (FPM) uses sequential multi-angle illumination and iterative phase retrieval to recover a high-resolution complex image from a series of low-resolution brightfield and darkfield images. We present OpenFPM, an open-source FPM platform in which conventional and optomechanical hardware is replaced with compact, low-cost 3D printed components. Illumination, sample and objective positioning, and camera triggering are controlled using a Python-based interface on a Raspberry Pi microcomputer. With a 10 x /0.25 NA objective lens and 636 nm illumination, OpenFPM experimentally achieves amplitude and phase reconstructions with an effective synthetic NA of 0.90 over a 1 mm field-of-view. This platform gives researchers accessible and affordable hardware for developing and testing LED-array microscopy techniques for a range of biomedical imaging applications.

Dragonfly and cicada wing-inspired titanium nanopillar surfaces show promising bactericidal properties for antibacterial medical implant applications, but the precise mechanisms of bacteria-nanopillar interactions under hydrated conditions remain unclear. Cryo-electron tomography (cryo-ET) enables the visualisation of cellular organelles within their native hydrated cellular environment at molecular resolution. Visualising the bacteria-material interface on nanostructured surfaces by cryo transmission electron microscopy (cryo-TEM) requires the preparation of thin lamellae. Obtaining lamellae of bacteria directly on metal substrates while in a non-fixed and hydrated state requires cryo-focused ion beam (cryo-FIB) milling to isolate the targeted bacteria from the bulk sample. This approach faces additional challenges compared to tissues or cells on TEM grids, as titanium samples require a simultaneous cross-section of soft and hard materials at the same position and require vitrification, which embeds the sample in a thick layer of ice. Nonetheless, we demonstrate how to target a specific bacterium interacting with a titanium nanopillar surface using correlative cryo-fluorescence imaging, and how lamellae can still be prepared from vitrified samples by extracting the targeted bacterium and its surrounding as a small volume and transferring it to a receptor grid for thin lamella preparation, called targeted cryo-lift-out. Here, we outline the workflows and discuss their advantages and limitations for producing lamellae through lift-out techniques under cryogenic conditions, using methods that do not involve gas injection systems (GIS) for the lift-out transfer. These advances enhance cryo-ET applications, enabling in situ investigations of the interface between bacteria and nanopillars to effectively study the bactericidal mechanisms of biomimetic nature-inspired nanotopographies in a hydrated environment.

The ability to detect host cell factors during Staphylococcus aureus infection in vitro by immunofluorescence microscopy is severely hampered by staphylococcal protein A (SpA), a cell wall-anchored protein that binds the fragment crystallizable (Fc) region of immunoglobulins. This interaction generates strong nonspecific fluorescent signals on the bacterial surface, complicating data interpretation and limiting the accuracy of quantitative image analysis. Several measures have been put forward to overcome this obstacle, most importantly the pre-incubation with an anti-SpA antibody (SpA) and the use of human serum (HS) as blocking agent and antibody diluent. To highlight this feature to general fluorescence microscopy users, we here systematically evaluated these two strategies. Using S. aureus coated on coverslips and S. aureus-infected A549 cells, we highlight the efficiencies of both approaches to markedly reduce nonspecific fluorescence, with HS treatment yielding the most profound suppression. Notably, HS, containing high levels of human immunoglobulins, offered a robust, cost-effective and broadly applicable solution for minimizing SpA-driven artifacts, thereby improving immunofluorescence microscopy in S. aureus infection models in vitro.

Digital holographic microscopy systems in a common-path configuration, compared to systems with a separate reference arm, offer a compact design and resistance to disturbances. They can operate with partially coherent illumination, reducing speckle noise. However, they are limited by the overlapping of the object beam and its laterally shifted replica. As a result, images from different regions of the object overlap on the detector, preventing imaging of dense samples. We present the wavelength-scanning replica-removal method, which solves this problem by enabling the separation of information from both replicas and thereby doubling the effective field of view (FOV). The wavelength-scanning multi-shear replica removal algorithm plays a key role in reconstructing the undisturbed phase from a series of holograms recorded with variable shears. The shear value is controlled by changing the illumination wavelength. This enabled the development of two measurement modes: time-domain wavelength scanning for high-quality imaging, and a single-shot mode with frame division into color channels to improve temporal resolution. The method was validated using resolution tests and biological samples - neurons and dynamic yeast cultures. By combining the advantages of the common-path configuration with dense-structure imaging and dynamic processes, the proposed method constitutes a versatile tool for quantitative phase microscopy.

Single-molecule localization microscopy (SMLM) methods enable fluorescence imaging of biological specimens with nanometer-scale resolution. Although fluorophore localization precision is theoretically limited only by photon statistics, in practice the resolution of SMLM images is often degraded by physical drift of the sample and/or the microscope during data acquisition. At present, correcting this effect requires either specialized stabilization systems or computationally intensive post-processing, and established drift correction algorithms based on image cross-correlation suffer from limited temporal resolution. In this study we introduce COMET, a new method for SMLM drift estimation which achieves a substantially higher precision, accuracy, and temporal resolution compared with existing algorithmic approaches. We demonstrate that improved drift estimation translates directly into higher SMLM image resolution, limited by localization precision rather than drift artifacts. COMET is applicable to all types of SMLM data, operating directly on 2D or 3D localization datasets, and is readily integrated into analysis workflows. We benchmark its performance using both simulations and experiments, including STORM, MINFLUX, and Sequential OligoSTORM measurements, where long acquisition times make drift correction particularly challenging. COMET is published as an open-source, Python-based software project and is also available on open cloud-computing platforms.

Multicolour and spectrally resolved single-molecule microscopy can reveal molecular interactions, nanoscale environments and dynamics, but usually depends on experimentally complex detection architectures based on beam splitting, spectral dispersion or engineered point spread functions. Here we show that spatially patterned detectors offer a conceptually simpler route to spectral single-molecule imaging. By replacing a conventional monochrome camera with a commercially available colour CMOS detector and fitting the raw detector response directly, we recover both molecular position and spectral fingerprint from a single image without optical splitting, channel registration or demosaicing. We term this approach spatial spectral single-molecule microscopy, or S3M. We show that S3M retains single-molecule sensitivity across the visible spectrum, enables robust spectral multiplexing, and supports applications spanning multicolour single-molecule tracking, single-molecule Forster resonance energy transfer, multicolour localisation microscopy and spectral PAINT. Although spatial patterning necessarily trades photon efficiency for spectral information, current low noise detectors already provide sufficient performance for a broad range of experiments. Spatially patterned detection therefore establishes a widely accessible strategy for simplified spectral microscopy and single-molecule spectroscopy, and points towards a new class of detector informed photonic measurement schemes for nanoscale imaging.

Backscattered electron scanning electron microscopy (BSE-SEM) provides compositional image contrast but has found limited application to biological samples due to the low atomic number difference between constituent elements, the thickness of the surrounding environment, and the need for complex sample preparation. Here, we demonstrate the use of room temperature liquid phase BSE-SEM (LPBSEM) for imaging Bacillus subtilis spores encapsulated in graphene liquid cells, preserving native hydration and reducing the thickness of the sample environment. This approach eliminates the need for staining and enables high-contrast visualisation of subcellular structures. Distinct structural layers within B. subtilis spores have been observed with a contrast similar to conventional thin-section transmission electron microscopy but without the need for sample preparation that potentially compromises sample integrity. We further investigate the influence of beam energy on the interaction volume depth and image contrast and propose optimal conditions for subsurface visualisation. Monte Carlo simulations have been used to validate our experimental observations and provide a quantitative framework for understanding BSE generation from hydrated, low atomic number specimens.

Quantitative measurements performed directly in vivo are necessary to understand how forces shape living tissues, yet this remains challenging due to optical scattering and mechanical complexity. Here, we present a method for making absolute force measurements using nanoscopic optical tweezers with a sensitivity of 300 fN in optically turbid biological media. Our approach combines back focal plane interferometry operating within the optical memory effect regime with a global fluctuation-dissipation fitting framework that simultaneously calibrates position detection, trap stiffness, and viscoelastic response. This method overcomes aberration-induced biases by jointly fitting passive fluctuations and driven harmonic responses, enabling robust force reconstruction in thick, scattering tissues within the mechanically relevant frequency range below 300 Hz. We validate our approach using highly scattering Drosophila pupae and embryos, demonstrating reliable in vivo measurements of forces and mechanical properties. Operating at a 1 kHz acquisition bandwidth, the system captures relevant mechanical dynamics without requiring extended high-frequency detection. Using this framework, we quantify the increase in cortical tension during pupal morphogenesis, characterize tissue viscoelasticity, and reveal stage-dependent variations in nuclear membrane tension during embryogenesis, even in the presence of strong ATP-driven fluctuations. Beyond bulk measurements, our method enables the quantitative mechanical characterization of single cells within mechanically coupled tissues.

Artificial vision systems hold transformative potential for biomedical imaging, diagnostics, and translational research by emulating and extending the capabilities of biological eyes. However, current techniques often face intrinsic trade-offs between spatial resolution, field of view, and depth perception, particularly in compact, biologically relevant settings. Here, we introduce FOLIC, a foveated light-field compound imaging system, which integrates compound-eye-inspired wide angular coverage and chambered-eye-inspired spatial acuity within a unified multi-aperture concave architecture. FOLIC naturally generates peripheral, blend, and foveated zones from a single capture, enabling seamless, depth-extended, multiscale visualization from wide-field context down to single-cell lateral resolution. We validate FOLIC across diverse fluorescent and non-fluorescent specimens, including cellular phantoms, tissue sections, and small organisms, demonstrating its versatility and scalability for biomedical research and related translational applications. We anticipate FOLIC to offer a biologically informed design blueprint for future artificial vision systems. TeaserA bioinspired system unifies compound and chambered eye principles to achieve wide-field volumetric microscopy.

Light sheet fluorescence microscopy (LSFM) is increasingly appreciated as the gold standard for gentle, volumetric imaging with fast acquisition speeds and/or long imaging durations. However, the often-constrained sample space of these microscopes has precluded a specific class of biological specimens from being studied with these tools: those requiring an air-liquid interface (ALI). Here, we present a device for robust imaging at ALI on an upright light sheet microscope with dipping objectives. We demonstrate the system using three relevant use-cases: ex vivo embryonic mouse salivary glands, human epidermal equivalent cultures, and in vivo adult Drosophila melanogaster brains. While the device presented is engineered for one specific light sheet microscope design, it provides a blueprint for easy adaptation to other systems. In doing so, it can potentially spur the use of LSFM for model systems that have so far been unable to take advantage of this powerful technology.

We present a live-to-cryo correlative imaging workflow for multiscale structural and chemical analysis of biological tissues in their near-native state. The method integrates live super-resolution fluorescence microscopy, live and cryogenic Raman spectroscopy, and targeted cryogenic focused ion beam/scanning electron microscopy, transmission electron microscopy, electron tomography, energy dispersive X-ray spectroscopy, and electron diffraction. This approach enables precise 3D targeting and nanoscale imaging of selected regions across four orders of magnitude in spatial resolution, while preserving ultrastructure and chemical composition. Using regenerating zebrafish scales as a benchmark, we visualize collagen fibril orientation, local matrix density, and mineral composition within the extracellular matrix. We identify a plywood-like architecture of unmineralized collagen with orientation-independent density variation, and reveal curved, acidic phosphate-rich mineral platelets aligned with collagen fibrils. This workflow establishes a generalizable strategy for comprehensive 3D correlative analysis of hybrid tissues, and opens new opportunities for studying native structure-function relationships at the interface of biology and materials science.

Ultraweak photon emission is the spontaneous emission of extremely low levels of light from a broad range of biological systems. Recent studies have reported that UPE measured extracranially can serve as a potential non-invasive biomarker of brain activity. Here, we show that this interpretation suffers from serious problems. First, when observed under properly dark conditions, the UPE from the head is much weaker than what is reported in certain papers on brain UPE from human heads. Signals detected in these studies are overwhelmingly dominated by background light. Second, photons at wavelengths < 600 nm are strongly attenuated by scalp and skull tissues, and longer wavelengths fall largely outside the effective spectral sensitivity of the photomultiplier tubes (PMTs) used. As a consequence, even if UPE from the head is detected under properly background-free conditions, it is likely to be dominated by emission from the scalp rather than from the brain, certainly as long as PMTs are used. Our results emphasize the importance of careful experimental design to make genuine progress on this important question.

Multimode fibers enable minimally invasive, high-resolution imaging through ultrathin probes, thereby enhancing diagnostic precision and facilitating real-time monitoring in delicate anatomical regions. In this work, we introduce HYFEN, a hydrogel-based endomicroscopic imaging platform for flexible, biocompatible, and subcellular-scale fluorescence microscopy. HYFEN leverages the unique properties of hydrogel materials, adaptive optics, and pixel-wise image enhancement to address challenges associated with silica-based fibers, including mode scrambling, limited field of view, and mechanical rigidity. The technique achieves precise mode threading, rapid diffraction-limited focusing at kilohertz speeds, and high-fidelity fluorescence signal acquisition with subcellular resolution. Notably, the approach extends fluorescence imaging under enhanced fiber dimensions and bending conditions that are unachievable with conventional modalities. Together, these advances establish HYFEN as a versatile platform for next-generation biointerfacing and minimally invasive imaging across biomedical and clinical settings.

Cryo-Focused Ion Beam Scanning Electron Microscopy (cryoFIB-SEM) using samples fixed by high-pressure freezing uniquely enables high resolution cryo-volume Electron Microscope (cvEM) images of cell ultrastructure to be obtained from whole cells and complex tissues in their near native state. As the freezing process also preserves fluorescence, the link between three-dimensional (3D) ultrastructure and biological process is also enabled by targeted cryo-Correlative Light and Electron Microscopy (CLEM). However, the overall viability of cvEM is challenged by sample preparation, charge balance during imaging, sample sensitivity to beam damage, contamination, and very long acquisition times. Here we detail new experimental workflows to significantly reduce each of these effects and demonstrate the improvement in resolution possible with results from the nematode Caenorhabditis elegans and the ciliated protozoon Paramecium bursaria containing many endosymbiotic algae. These results demonstrate the versatility and potential wide-ranging utility of cvEM for 3D ultrastructural imaging of whole multicellular and unicellular organisms.