Abstract 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.

Nanoparticle capsules (NCs), capsule-like structures composed of inorganic nanoparticles (NPs), hold great potential for diverse applications due to their structure-driven functionality and the unique properties of the NPs. Despite advancements in NC formation methods, achieving both stable formation and efficient material encapsulation remains challenging. In this study, a simple and versatile method is developed for the formation of inorganic NCs. Inorganic NCs are formed from oligo(ethylene glycol) (OEG)-modified inorganic NPs via self-assembly at solute-induced liquid-liquid interfaces. First, Au NCs are formed from OEG-Au NPs. Their formation is confirmed by scanning transmission electron microscopic (STEM) and cryo-electron tomographic (ET) observations. The resulting Au NCs exhibited high stability, uniform size, and precise tunability under various conditions. The method is further extended to magnetic Fe3O4 NPs, affording size-controlled magnetic NCs, demonstrating broad applicability. Moreover, this approach enables the efficient encapsulation of various materials, including citrate-capped Au NPs and DNA molecules, into inorganic NCs, affording >2000-fold enrichment. This method paves the way for innovative application of inorganic NCs in drug delivery, nanoreactors, and beyond.

Metal nanoparticle assemblies induce interparticle plasmonic coupling, and precise control of this coupling by arranging the geometrical and spatial conditions is crucial for the application of plasmonic materials. In this study, gold nanodiscs (AuNDs) modified with complementary short DNA ligands (positive-sense DNA, (+)-DNA; negative-sense DNA, (-)-DNA) were assembled into 1D cylindrical structures with various interparticle gap distances and degrees of multimerization, enabling the precise tuning of plasmonic coupling strength. Specifically, AuNDs (90 nm in diameter) functionalized with 11-base (+)-DNA and (-)-DNA ligands were mixed and assembled into multimers through face-to-face interactions, driven by the hybridization of short DNA ligands without linker strands. This assembly induced strong plasmonic coupling owing to the short interparticle gap distance (5.7 nm), which led to significant peak shifts in the extinction spectra. Additionally, the plasmonic coupling strength was fine-tuned by varying the interparticle gap distance using DNA strands of different lengths (8, 11, and 20 base pairs). Furthermore, the assembly and disassembly temperatures of the DNA-AuNDs were tuned by adjusting (i) the salt concentration in the solution and (ii) the length of the DNA ligands. Finally, the degree of multimerization, which has a pronounced effect on the plasmonic coupling strength, was modulated by adjusting (i) the molar ratio of (+)-DNA-AuNDs to (-)-DNA-AuNDs and (ii) the salt concentration in the solution. This paper presents a simple and effective method to tune the plasmonic coupling of AuND assemblies, thereby expanding the potential applications of plasmonic materials.

We report that complexes formed between gold nanorods (AuNRs) and metal-mediated DNA exhibit plasmonic circular dichroism (CD) signals up to ∼400 times stronger than the molecular CD signal of DNA. This substantial enhancement enables the detection of metal ions, offering a promising approach to analytical applications in chiral biochemistry.

Abstract Gold nanorods (AuNRs) have unique optical properties such as transverse and longitudinal localized surface plasmon resonance (T‐ and L‐LSPR). As the L‐LSPR absorption depends on the angle of the AuNRs to incident light and polarization, orientational control of AuNRs is a crucial issue. In spite of various techniques to control AuNR orientation, dynamic orientation tuning on a solid substrate remains challenging. Herein, dynamic changes are demonstrated in AuNR orientation in the anionic polymer (DNA) brushes via control of their thickness by salt concentration. AuNRs vertically align toward the substrates when their thickness exceeds the AuNR length. Once their thickness becomes shorter than the AuNR length, the attached AuNRs begin to tilt. The tilt reaches a maximum level close to horizontal when the thickness decreases to half that of the AuNR length. The dynamic control between the vertical (uniform) and tilted (random) orientation of the AuNRs showed not only absorption intensity changes in L‐LSPR but also the switching of side‐by‐side plasmon coupling. The polymer brush‐based system affords a novel platform for the stimuli‐responsive control of AuNR orientation on the substrates via changes in the thickness of polymer brushes for actively tunable plasmonic substrates.

Surface-enhanced Raman scattering (SERS) is an attractive technique in molecular detection with high sensitivity and label-free characteristics. However, its use in protein detection is limited by the large volume of proteins, hindering its approach to the narrow spaces of hotspots. In this study, we fabricated a Au nanoTriangle plate Array on Gel (AuTAG) as an SERS substrate by attaching a Au nanoTriangle plate (AuNT) arrangement on a thermoresponsive hydrogel surface. The AuTAG acts as an actively tunable plasmonic device, on which the interparticle distance is altered by controlling temperature via changes in hydrogel volume. Further, we designed a Gel Filter Trapping (GFT) method as an active protein delivery strategy based on the characteristics of hydrogels, which can absorb water and separate biopolymers through their three-dimensional (3D) polymer networks. On the AuTAGs, fabricated with AuNTs modified with charged surface ligands to prevent the nonspecific adsorption of analytes to particles, the GFT method helped the delivery of proteins to hotspot areas on the AuNT arrangement. This combination of a AuTAG substrate and the GFT method enables ultrahigh sensitivity for protein detection by SERS up to a single-molecule level as well as a wide quantification concentration range of 6 orders due to their geometric advantages.

The emerging concept of tunable plasmonicchirality is mostly observed as a reconfigurable behavior or afeature of complex chiral plasmonic assemblies. For discretecolloidal particles, it is challenging to achieve reversible tunabilityor a transient response with regard to chiroptical activities,particularly in the visible or near-infrared region. Herein, wedemonstrate a stimulus-responsive system based on chiralmolecule-achiral plasmonic nanoparticles coated with polyaniline(PANI) as a variable dielectric layer, in whichL-/D-cysteinemolecules are introduced between the gold core and the shell as astatic chiral source, allowing the chiral transfer effect to be greatlyamplified by the hotspot gap of sub-monomolecular thickness. By taking advantage of the responsive properties to either pH or theelectric potential dual stimuli of PANI, which also provides a stable and real-time switchable dielectric environment for the wholesystem, dynamic tuning of the plasmon and its induced chiroptical activities of core-gap-shell nanoparticles@PANI were preciselyobtained. This well-defined design provides an open platform forflexible and rational tailoring of plasmonic cores, chiral molecules,and variable dielectrics to chiroptical needs, which is important for realizing applications in chemical sensing, chiral nanocatalysis,enantioselective separations, and novel optical devices

Anisotropic gold nanodiscs (AuNDs) possess unique properties, such as large flat surfaces and dipolar plasmon modes, which are ideal constituents for the fabrication of plasmonic assemblies for novel and emergent functions. In this report, we present the thermo-responsive assembly and thermo-dynamic behavior of AuNDs functionalized with methyl-hexa(ethylene glycol) undecane-thiol as a thermo-responsive ligand. Upon heating, the temperature stimulus caused a blue shift of the plasmon peak to form a face-to-face assembly of AuNDs due to the strong hydrophobic and van der Waals interactions between their large flat surfaces. Importantly, AuNDs allowed for the incorporation of the carboxylic acid-terminated ligand while maintaining their thermo-responsive assembly ability. With regard to their reversible assembly/disassembly behavior in the thermal cycling process, significant rate-independent hysteresis, which is related to their thermo-dynamics, was observed and was shown to be dependent on the carboxylic acid content of the surface ligands. As AuNDs have not only unique plasmonic properties but also high potential for attachment due to the fact of their flat surfaces, this study paves the way for the exploitation of AuNDs in the development of novel functional materials with a wide range of applications.

Assemblies of cationic gold nanorods (AuNRs) via electrostatic interactions with double-stranded (ds) DNA were investigated in solution and after evaporation as a cast-film. Interestingly, moderately positively charged AuNRs assembled with dsDNA provided a monolayer sheet with an ordered alignment resembling a two-dimensional (2D) smectic structure during solvent evaporation.

Biomolecular systems actively control their local environment on a sub-nm scale via changes in molecular configuration from their flexible structures and derive emergent functions. Although this functional emergence based on local environmental control is attracting a great deal of attention in chemistry, it remains challenging to realize this artificially. Herein, we report the tuning of the thermo-responsive properties of oligo(ethylene glycol) (OEG) derivatives attached on gold nanoparticles via local environmental control not only by the hydrophobic moiety at their terminus but also by their molecular configuration. OEG-attached alkane thiol-modified AuNPs showed thermo-responsive assembly/disassembly in water through the hydration/dehydration of the OEG portions in a manner dependent both on the hydrophobicity at their terminus and the surface curvature of the core nanoparticles. Further, the assembly temperature (T-A) was also tuned by ligand mixing with a non-thermo-responsive ligand with a shorter OEG length. Molecular dynamics simulations show that the distribution of the hydrophobic terminus in the normal direction along the gold surface varied in accordance with the surface curvature, indicating variations in molecular configuration. It is expected that a bent configuration could accelerate the thermo-responsiveness of OEG by allowing them greater accessibility to the hydrophobic terminus. Experimental and simulation results support the notion that local OEG density tuning by surface curvature or ligand mixing with a different OEG length leads to different degrees of accessibility to the hydrophobic terminus via changes in molecular configuration, promoting local environmental control-directed assembly temperature tuning.

Phototrophs assimilate CO2 into organic compounds that accumulate in storage organelles. Elucidation of the carbon dynamics of storage organelles could enhance the production efficiency of valuable compounds and facilitate the screening of strains with high photosynthetic activity. To comprehensively elucidate the carbon dynamics of these organelles, the intraorganellar distribution of the carbon atoms that accumulate at specific time periods should be probed. In this study, the biosynthesis of polysaccharides in storage organelles was spatiotemporally probed via stimulated Raman scattering (SRS) microscopy using a stable isotope (13C) as the tracking probe. Paramylon granules (a storage organelle of β-1,3-glucan) accumulated in a unicellular photosynthetic alga, Euglena gracilis, were investigated as a model organelle. The carbon source of the culture medium was switched from NaH12CO3 to NaH13CO3 during the production of the paramylon granules; this resulted in the distribution of the 12C and 13C constituents in the granules, so that the biosynthetic process could be tracked. Taking advantage of high-resolution SRS imaging and label switching, the localization of the 12C and 13C constituents inside a single paramylon granule could be visualized in three dimensions, thus revealing the growth process of paramylon granules. We propose that this method can be used for comprehensive elucidation of the dynamic activities of storage organelles.

Cellular metabolites are valuable in a diverse range of applications. For example, the unicellular green alga Haematococcus lacustris produces as a secondary metabolite the carotenoid pigment astaxanthin (AXT), which is widely used in nutraceutical, cosmetic, and food industries due to its strong antioxidant activity. In order to enhance the productivity of H. lacustris, spatial and temporal understanding of its metabolic dynamics is essential. Here we show spatiotemporal monitoring of AXT production in H. lacustris cells by resonance Raman microscopy combined with stable isotope labeling. Specifically, we incorporated carbon dioxide (13CO2) labeled with a stable isotope (13C) into H. lacustris cells through carbon fixation and traced its conversion to 13C-AXT using our resonance Raman microscope. We incubated H. lacustris cells under various conditions by switching, pulsing, and replacing 13CO2 and 12CO2. By measurement of these cells we determined the fixation time of 13C-carbon, visualized the intracellular localization of 13C- and 12C-AXTs, and revealed the dynamic consumption-production equilibrium of the accumulated AXT. This work is a valuable step in the development of effective screening criteria for high AXT-producing H. lacustris cells.

In this study, we demonstrate that the plasmonic properties of gold nanorods (GNRs) electrostatically adsorbed on a DNA brush substrate are reversibly controlled by changes in NaCl concentration. This plasmonic change results from GNR assembly/disassembly in a DNA brush layer. In addition, we show that this active plasmonic system exhibits intense and switchable chiroptical properties.

Nanoparticles exhibit a number of unique properties such as localized surface plasmon resonance (LSPR). As this LSPR is sensitive to geometrical or spatial conditions, the arrangement of nanoparticles, in particular the active arrangement of plasmonic structures, is an important issue. In this study, gold nanorod (GNR) arrays were prepared by GNR attachment on anionic polymer (DNA) brushes via electrostatic interactions and their stimuli-responsive changes in orientationwere investigated. As a result, the orientation of GNR arrays on DNA brushes reversibly changed by the modulation of electrostatic interactions between GNRs and polymers via changes in the solution pH. As these extensive GNR arrays are prepared via easy bottom-up processes, GNR surface properties are easily tuned by simple modification, and DNAs could be replaced with various synthetic polymers, we believe that this study will lead to the development of nextgeneration materials and devices with actively tunable structures.

Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm-1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris.

Time-course analysis of single cells is important to characterize heterogeneous activities of individual cells such as the metabolic response to their environment. Single-cell isolation is an essential step prior to time-course analysis of individual cells by collecting, culturing, and identifying multiple single-cell targets. Although single-cell isolation has been performed by various methods previously, a glass microfluidic device with semiclosed microchannels dramatically improved this process with its simple operation and easy transfer for time-course analysis of identified single cells. This study demonstrates isolating single cells of the highly motile microalgae, Euglena gracilis, by semiclosed microchannels with liquid flow only. The isolated single cells were identified in isolating channels and continuously cultured to track, by Raman microscopy, for the formation of subcellular granules composed of polysaccharide paramylon, a unique metabolite of E. gracilis, generated through photosynthesis. Through low-temperature glass bonding, a thin glass interface was incorporated to the microfluidic device. Thus, the device could perform the direct measurements of cultured single cells at high magnification by Raman microscopy with low background noise. In this study, the first demonstration of sequential monitoring of paramylon biogenesis in a single identified E. gracilis cell is shown.

Abiotic ligands that bind to specific biomolecules have attracted attention as substitutes for biomolecular ligands, such as antibodies and aptamers. Radical polymerization enables the production of robust polymeric ligands from inexpensive functional monomers. However, little has been reported about the production of monodispersed polymeric ligands. Herein, we present homogeneous ligands prepared via radical polymerization that recognize epitope sequences on a target peptide and neutralize the toxicity of the peptide. Taking advantage of controlled radical polymerization and separation, a library of multifunctional oligomers with discrete numbers of functional groups was prepared. Affinity screening revealed that the sequence specificity of the oligomer ligands strongly depended on the number of functional groups. The process reported here will become a general step for the development of abiotic ligands that recognize specific peptide sequences.