3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway.

Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.

On-the-fly Raman microscopy guaranteeing the accuracy of discrimination.

Accelerating the measurement for discrimination of samples, such as classification of cell phenotype, is crucial when faced with significant time and cost constraints. Spontaneous Raman microscopy offers label-free, rich chemical information but suffers from long acquisition time due to extremely small scattering cross-sections. One possible approach to accelerate the measurement is by measuring necessary parts with a suitable number of illumination points. However, how to design these points during measurement remains a challenge. To address this, we developed an imaging technique based on a reinforcement learning in machine learning (ML). This ML approach adaptively feeds back "optimal" illumination pattern during the measurement to detect the existence of specific characteristics of interest, allowing faster measurements while guaranteeing discrimination accuracy. Using a set of Raman images of human follicular thyroid and follicular thyroid carcinoma cells, we showed that our technique requires 3,333 to 31,683 times smaller number of illuminations for discriminating the phenotypes than raster scanning. To quantitatively evaluate the number of illuminations depending on the requisite discrimination accuracy, we prepared a set of polymer bead mixture samples to model anomalous and normal tissues. We then applied a home-built programmable-illumination microscope equipped with our algorithm, and confirmed that the system can discriminate the sample conditions with 104 to 4,350 times smaller number of illuminations compared to standard point illumination Raman microscopy. The proposed algorithm can be applied to other types of microscopy that can control measurement condition on the fly, offering an approach for the acceleration of accurate measurements in various applications including medical diagnosis.

Subcellular localization of Type VI secretion system assembly in response to cell-cell contact.

Bacteria require a number of systems, including the type VI secretion system (T6SS), for interbacterial competition and pathogenesis. The T6SS is a large nanomachine that can deliver toxins directly across membranes of proximal target cells. Since major reassembly of T6SS is necessary after each secretion event, accurate timing and localization of T6SS assembly can lower the cost of protein translocation. Although critically important, mechanisms underlying spatiotemporal regulation of T6SS assembly remain poorly understood. Here, we used super-resolution live-cell imaging to show that while Acinetobacter and Burkholderia thailandensis can assemble T6SS at any site, a significant subset of T6SS assemblies localizes precisely to the site of contact between neighboring bacteria. We identified a class of diverse, previously uncharacterized, periplasmic proteins required for this dynamic localization of T6SS to cell-cell contact (TslA). This precise localization is also dependent on the outer membrane porin OmpA. Our analysis links transmembrane communication to accurate timing and localization of T6SS assembly as well as uncovers a pathway allowing bacterial cells to respond to cell-cell contact during interbacterial competition.

Optical single-pixel volumetric imaging by three-dimensional light-field illumination.

Three-dimensional single-pixel imaging (3D SPI) has become an attractive imaging modality for both biomedical research and optical sensing. 3D-SPI techniques generally depend on time-of-flight or stereovision principle to extract depth information from backscattered light. However, existing implementations for these two optical schemes are limited to surface mapping of 3D objects at depth resolutions, at best, at the millimeter level. Here, we report 3D light-field illumination single-pixel microscopy (3D-LFI-SPM) that enables volumetric imaging of microscopic objects with a near-diffraction-limit 3D optical resolution. Aimed at 3D space reconstruction, 3D-LFI-SPM optically samples the 3D Fourier spectrum by combining 3D structured light-field illumination with single-element intensity detection. We build a 3D-LFI-SPM prototype that provides an imaging volume of ∼390 × 390 × 3,800 µm3 and achieves 2.7-µm lateral resolution and better than 37-µm axial resolution. Its capability of 3D visualization of label-free optical absorption contrast is demonstrated by imaging single algal cells in vivo. Our approach opens broad perspectives for 3D SPI with potential applications in various fields, such as biomedical functional imaging.

DNA damage reduces heterogeneity and coherence of chromatin motions.

Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.

State transition is quiet around pyrenoid and LHCII phosphorylation is not essential for thylakoid deformation in Chlamydomonas 137c.

Photosynthetic organisms have developed a regulation mechanism called state transition (ST) to rapidly adjust the excitation balance between the two photosystems by light-harvesting complex II (LHCII) movement. Though many researchers have assumed coupling of the dynamic transformations of the thylakoid membrane with ST, evidence of that remains elusive. To clarify the above-mentioned coupling in a model organism Chlamydomonas, here we used two advanced microscope techniques, the excitation-spectral microscope (ESM) developed recently by us and the superresolution imaging based on structured-illumination microscopy (SIM). The ESM observation revealed ST-dependent spectral changes upon repeated ST inductions. Surprisingly, it clarified a less significant ST occurrence in the region surrounding the pyrenoid, which is a subcellular compartment specialized for the carbon-fixation reaction, than that in the other domains. Further, we found a species dependence of this phenomenon: 137c strain showed the significant intracellular inhomogeneity of ST occurrence, whereas 4A+ strain hardly did. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations caused by the ST-inducing illumination. This fine, irreversible thylakoid transformation was also observed in the STT7 kinase-lacking mutant. This result revealed that the fine thylakoid transformation is not induced solely by the LHCII phosphorylation, suggesting the highly susceptible nature of the thylakoid ultrastructure to the photosynthetic light reactions.

Far-Field Phase-Shifting Structured Light Illumination Enabled by Polarization Multiplexing Metasurface for Super-Resolution Imaging.

The phase-shifting structured light illumination technique is widely used in imaging but often relies on mechanical translation stages or spatial light modulators, leading to system instability, low displacement accuracy, and limited integration feasibility. In response to these challenges, we propose and demonstrate an approach for generating far-field phase-shifting structured light using a polarization multiplexing metasurface. By controlling the polarization states of incident and transmitted light, the metasurface creates a three-step displacement of structured light, eliminating the need to move samples or illumination sources. As a proof of concept, we experimentally demonstrate microscopic imaging using structured light illumination generated by metasurfaces, extracting high-frequency information from objects, and surpassing the diffraction limit. The proposed metasurface platform offers a promising approach for developing compact and robust phase-shifting imaging systems, with broad prospects in quantitative detection, machine vision, and beyond.

Localized Plasmonic Structured Illumination Microscopy Using Hybrid Inverse Design.

Super-resolution fluorescence imaging has offered unprecedented insights and revolutionized our understanding of biology. In particular, localized plasmonic structured illumination microscopy (LPSIM) achieves video-rate super-resolution imaging with ∼50 nm spatial resolution by leveraging subdiffraction-limited nearfield patterns generated by plasmonic nanoantenna arrays. However, the conventional trial-and-error design process for LPSIM arrays is time-consuming and computationally intensive, limiting the exploration of optimal designs. Here, we propose a hybrid inverse design framework combining deep learning and genetic algorithms to refine LPSIM arrays. A population of designs is evaluated using a trained convolutional neural network, and a multiobjective optimization method optimizes them through iteration and evolution. Simulations demonstrate that the optimized LPSIM substrate surpasses traditional substrates, exhibiting higher reconstruction accuracy, robustness against noise, and increased tolerance for fewer measurements. This framework not only proves the efficacy of inverse design for tailoring LPSIM substrates but also opens avenues for exploring new plasmonic nanostructures in imaging applications.

Synergism in Binary Nanocrystals Enables Top-Illuminated HgTe Colloidal Quantum Dot Short-Wave Infrared Imager.

Thanks to their tunable infrared absorption, solution processability, and low fabrication costs, HgTe colloidal quantum dots (CQDs) are promising for optoelectronic devices. Despite advancements in device design, their potential for imaging applications remains underexplored. For integration with Si-based readout integrated circuits (ROICs), top illumination is necessary for simultaneous light absorption and signal acquisition. However, most high-performing traditional HgTe CQD photodiodes are p-on-n stack and bottom-illuminated. Herein, we report top-illuminated inverted n-on-p HgTe CQD photodiodes using a robust p-type CQD layer and a thermally evaporated Bi2S3 electron transport layer. The p-type CQD solid is achieved by exploring the synergism in binary HgTe and Ag2Te CQDs. These photodetectors show a room-temperature detectivity of 3.4 × 1011 jones and an EQE of ∼44% at ∼1.7 µm wavelength, comparable to the p-on-n HgTe CQD photodiodes. A top-illuminated HgTe CQD short-wave infrared imager (640 × 512 pixels) was fabricated, demonstrating successful infrared imaging.

Helical chromonema coiling is conserved in eukaryotes.

Efficient chromatin condensation is required to transport chromosomes during mitosis and meiosis, forming daughter cells. While it is well accepted that these processes follow fundamental rules, there has been a controversial debate for more than 140 years on whether the higher-order chromatin organization in chromosomes is evolutionarily conserved. Here, we summarize historical and recent investigations based on classical and modern methods. In particular, classical light microscopy observations based on living, fixed, and treated chromosomes covering a wide range of plant and animal species, and even in single-cell eukaryotes suggest that the chromatids of large chromosomes are formed by a coiled chromatin thread, named the chromonema. More recently, these findings were confirmed by electron and super-resolution microscopy, oligo-FISH, molecular interaction data, and polymer simulation. Altogether, we describe common and divergent features of coiled chromonemata in different species. We hypothesize that chromonema coiling in large chromosomes is a fundamental feature established early during the evolution of eukaryotes to handle increasing genome sizes.

Identification of z-axis filopodia in growth cones using super-resolution microscopy.

A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three-dimensional-structured illumination microscopy, a type of super-resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z-axis ("z-filopodia") within the growth cone. Z-filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z-filopodia formation and dynamics were regulated by actin-regulatory proteins, such as vasodilator-stimulated phosphoprotein, fascin, and cofilin. Chromophore-assisted laser inactivation of cofilin induced the rapid turnover of z-filopodia. An axon guidance receptor, neuropilin-1, was concentrated in z-filopodia and was transported together with them, whereas its ligand, semaphorin-3A, was selectively bound to them. Membrane domains associated with z-filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin-1. The results suggest that z-filopodia have unique turnover properties, and unlike xy-filopodia, do not function as force-generating structures for axon extension.

Quantum illumination and quantum radar: a brief overview.

Quantum illumination (QI) and quantum radar have emerged as potentially groundbreaking technologies, leveraging the principles of quantum mechanics to revolutionise the field of remote sensing and target detection. The protocol, particularly in the context of quantum radar, has been subject to a great deal of aspirational conjecture as well as criticism with respect to its realistic potential. In this review, we present a broad overview of the field of quantum target detection focusing on QI and its potential as an underlying scheme for a quantum radar operating at microwave frequencies. We provide context for the field by considering its historical development and fundamental principles. Our aim is to provide a balanced discussion on the state of theoretical and experimental progress towards realising a working QI-based quantum radar, and draw conclusions about its current outlook and future directions.

Super-resolution microscopy reveals the number and distribution of topoisomerase IIα and CENH3 molecules within barley metaphase chromosomes.

Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination, respectively. Consequently, they are required for proper chromosome segregation during cell divisions. We combined two super-resolution techniques, structured illumination microscopy (SIM) to co-localize Topo IIα and CENH3, and photoactivated localization microscopy (PALM) to determine their molecule numbers in barley metaphase chromosomes. We detected a dispersed Topo IIα distribution along chromosome arms but an accumulation at centromeres, telomeres, and nucleolus-organizing regions. With a precision of 10-50 nm, we counted ~ 20,000-40,000 Topo IIα molecules per chromosome, 28% of them within the (peri)centromere. With similar precision, we identified ~13,500 CENH3 molecules per centromere where Topo IIα proteins and CENH3-containing chromatin intermingle. In short, we demonstrate PALM as a useful method to count and localize single molecules with high precision within chromosomes. The ultrastructural distribution and the detected amount of Topo IIα and CENH3 are instrumental for a better understanding of their functions during chromatin condensation and centromere determination.

Multiview tiling light sheet microscopy for 3D high-resolution live imaging.

Light-sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.

Photo-Excited High-Spin State Ni (III) Species in Mo-Doped Ni3S2 for Efficient Urea Oxidation Reaction.

Designing robust catalysts for increasing the sluggish kinetics of the urea oxidation reaction (UOR) is challenging. Herein, the regulation of spin states for metal active sites by photoexcitation to facilitate the adsorption of urea and intermediates is demonstrated. Mo-doped nickel sulfide nanoribbon arrays (Mo-Ni3S2@NMF) with excellent light-trapping capacity are successfully prepared. Under AM 1.5G illumination, the activity of the Mo-Ni3S2@NMF exhibits a 50% improvement in the UOR current. Compared with those under dark conditions, Mo-Ni3S2@NMF achieve 10 mA cm-2 at 1.315 VRHE for UOR and 1.32 Vcell for urea electrolysis, which are decreases of 15 and 80 mV, respectively. The electron spin resonance, in situ Fourier transform infrared spectroscopy analysis and density functional theory calculations reveal that illumination led to the formation of Ni3+ active sites in a high-spin state, which strengthens the d-p orbital hybridization of Ni-N, hence facilitating the adsorption of urea. C─N cleavage of the *CONN intermediate is further inhibited, which promotes the oxidation of urea molecules via the active N2 pathway, thereby accelerating the UOR rate.

Biochemical versus stomatal acclimation of dynamic photosynthetic gas exchange to elevated CO2 in three horticultural species with contrasting stomatal morphology.

Understanding photosynthetic acclimation to elevated CO2 (eCO2) is important for predicting plant physiology and optimizing management decisions under global climate change, but is underexplored in important horticultural crops. We grew three crops differing in stomatal density-namely chrysanthemum, tomato, and cucumber-at near-ambient CO2 (450 µmol mol-1) and eCO2 (900 µmol mol-1) for 6 weeks. Steady-state and dynamic photosynthetic and stomatal conductance (gs) responses were quantified by gas exchange measurements. Opening and closure of individual stomata were imaged in situ, using a novel custom-made microscope. The three crop species acclimated to eCO2 with very different strategies: Cucumber (with the highest stomatal density) acclimated to eCO2 mostly via dynamic gs responses, whereas chrysanthemum (with the lowest stomatal density) acclimated to eCO2 mostly via photosynthetic biochemistry. Tomato exhibited acclimation in both photosynthesis and gs kinetics. eCO2 acclimation in individual stomatal pore movement increased rates of pore aperture changes in chrysanthemum, but such acclimation responses resulted in no changes in gs responses. Although eCO2 acclimation occurred in all three crops, photosynthesis under fluctuating irradiance was hardly affected. Our study stresses the importance of quantifying eCO2 acclimatory responses at different integration levels to understand photosynthetic performance under future eCO2 environments.

Modification of the electrokinetic motion of microalgae through light illumination for viability assessment.

The viability detection of microalgae with the electrokinetic (EK) technique shows vast applications in the biology and maritime industry. However, due to the slight variations in the EK properties between alive and dead microalgae cells, the accuracy and practicability of this technique is limited. In this paper, the light illumination pretreatment was conducted to modify the EK velocity of microalgae for enhancing the EK difference. The effects of the illumination time and light color on the EK velocities of Chlorella vulgaris and Isochrysis galbana were systematically measured, and the EK differences between alive and dead cells were calculated and compared. The results indicate that under light illumination, the photosynthesis of the alive cells leads to the amplification of the zeta potential, leading toward increase in the EK difference along with the illumination time. By using light with different color spectra to treat the microalgae, it was found that the EK difference changes with the light color according to the following order: white light > red light > blue light > green light. The difference in EK potential with exposure to white light treatment surpasses over 10-fold in comparison to those without such treatment. The light pretreatment technique, as illustrated in this study, offers an advantageous strategy to enhance the EK difference between living and dead cells, proving beneficial in the field of microalgae biotechnology.

In vivo biosensing of subcellular pyruvate pools reveals photosynthesis-dependent metabolite dynamics in Nicotiana benthamiana.

Pyruvate is central to metabolism across biology. It acts as a metabolic hub linking key pathways including glycolysis, the Krebs cycle, fermentation, and synthesis of amino acids, fatty acids, isoprenoids and nucleotides. Even though the central role of pyruvate is well established biochemically, there is a remarkable void in our understanding of how pyruvate levels behave within cells, where pyruvate is distributed across different compartments, and differential changes in pyruvate pools may occur rapidly upon changes in metabolic fluxes. Recently, this problem has been addressed by the development of a genetically-encoded pyruvate biosensor to provide first insights into the pyruvate dynamics in animal cells. Here, we establish in vivo biosensing of pyruvate in plants. We provide advanced characterisation of the biosensor properties and demonstrate the functionality of the sensor in the cytosol, the mitochondria and the chloroplasts of Nicotiana benthamiana epidermal cells. Finally, we harnessed the tool to investigate the impact of photosynthesis on pyruvate with unprecedented spatial and temporal resolution, revealing pronounced changes in cytosolic pyruvate pools. While highlighting the current limitations of the biosensor, this study provides proof-of-concept for how the dynamics and regulation of central carbon metabolites can be revealed in the context of living plant tissues.

Effect of Photoperiod and Illuminance on Daily Activity Patterns, Physiology, and NPY Peptide Expression in Migratory Redheaded Buntings (Emberiza bruniceps).

INTRODUCTION: Light is the primary source of energy and regulates seasonal changes in physiology and behavior. The role of photoperiod has been much investigated in several bird species, but the role of illumination in seasonal adaptations of passerine finches is less understood. We, therefore, investigated the effects of photoperiod and illuminance on migratory physiology in a Palearctic-Indian migratory finch, redheaded bunting (Emberiza bruncieps). METHODS: Photosensitive buntings maintained under short days (8L:16D) were divided into three groups receiving 5, 25, and 100 lux of white daytime illuminance, respectively. Thereafter, using photoperiodic manipulation three life history states, i.e., nonmigratory (NM), premigratory (PM), and migratory (MIG) states were induced in the buntings. The birds in the MIG state were consecutively perfused after seven nights of Zugunruhe (nighttime migratory restlessness) for neuropeptide Y (NPY)-immunohistochemistry, which is involved in a wide range of functions including energy homeostasis, vision, and fat deposition in birds. RESULTS: We found differential effects of illuminance on locomotor activity and physiology. Photostimulated birds showed intense nighttime activity in the MIG state. We observed premigratory hyperphagia in the birds, with increased food intake in the 100 lux group, which was reflected in the body mass gain in the MIG state. NPY expression on the periphery of the nucleus rotundus suggests its potential role in visual acuity, where the NPY-cell count significantly decreased under 25 lux illumination. CONCLUSION: We demonstrate that migrating birds may also experience physiological effects from changes in daytime illumination. We observed illuminance-dependent variations in the quantity of food consumed by the birds. It indicates that the illuminance may also impact the encephalic centers that control food intake.

Modulated illumination microscopy: Application perspectives in nuclear nanostructure analysis.

The structure of the cell nucleus of higher organisms has become a major topic of advanced light microscopy. So far, a variety of methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well as different types of patterned illumination microscopy, modulated either laterally (in the object plane) or axially (along the optical axis). Based on our experience, we discuss here some application perspectives of Modulated Illumination Microscopy (MIM) and its combination with single-molecule localisation microscopy (SMLM). For example, spatially modulated illumination microscopy/SMI (illumination modulation along the optical axis) has been used to determine the axial extension (size) of small, optically isolated fluorescent objects between ≤ 200 nm and ≥ 40 nm diameter with a precision down to the few nm range; it also allows the axial positioning of such structures down to the 1 nm scale; combined with laterally structured illumination/SIM, a 3D localisation precision of ≤1 nm is expected using fluorescence yields typical for SMLM applications. Together with the nanosizing capability of SMI, this can be used to analyse macromolecular nuclear complexes with a resolution approaching that of cryoelectron microscopy.