Publications

Tissue engineering holds great promise for biomedical research and healthcare, offering alternatives to animal models and enabling tissue regeneration and organ transplantation. Three-dimensional (3D) bioprinting stands out for its design flexibility and reproducibility. Here, we present an integrated fluorescent light sheet bioprinting and imaging system that combines high printing speed (0.66 mm3 /s) and resolution (9 μm) with light sheet-based imaging. This approach employs direct laser patterning and a static light sheet for confined voxel crosslinking in photocrosslinkable materials. The developed bioprinter enables real-time monitoring of hydrogel crosslinking using fluorescent recovery after photobleaching (FRAP) and brightfield imaging as well as in situ light sheet imaging of cells. Human fibroblasts encapsulated in a thiol-ene click chemistry-based hydrogel exhibited high viability (83% ± 4.34%) and functionality. Furthermore, full-thickness skin constructs displayed characteristics of both epidermal and dermal layers and remained viable for 41 days. The integrated approach demonstrates the capabilities of light sheet bioprinting, offering high speed, resolution, and real-time characterization. Future enhancements involving solid-state laser scanning devices such as acousto-optic deflectors and modulators will further enhance resolution and speed, opening new opportunities in light-based bioprinting and advancing tissue engineering. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

JTD Keywords: cadherin, collagen, culture, differentiation, fluorescence microscopy, full-thickness skin model, hydrogels, light sheet bioprinter, light sheet fluorescence microscopy, proliferation, survival, tissue engineering, Animal, Animals, Biofabrication, Bioprinting, Cell culture, Crosslinking, Fluorescence, Fluorescence microscopy, Full-thickness skin model, Hair follicle, Human, Humans, Hydrogel, Hydrogels, Image resolution, Laser patterning, Light sheet, Light sheet bioprinter, Light sheet fluorescence microscopy, Molecular biology, Photobleaching, Printing, three-dimensional, Procedures, Reproducibility, Reproducibility of results, Skin model, Three dimensional printing, Tissue, Tissue engineering, Tissue regeneration, Tissue scaffolds, Tissues engineerings

Exposure of experimental rodents to controlled cycles of light, food, and temperature is important when investigating alterations in circadian cycles that profoundly influence health and disease. However, applying such stimuli simultaneously is difficult in practice. We aimed to design, build, test, and open-source describe a simple device that subjects a conventional mouse cage to independent cycles of physiologically relevant environmental variables. The device is based on a box enclosing the rodent cage to modify the light, feeding, and temperature environments. The device provides temperature-controlled air conditioning (heating or cooling) by a Peltier module and includes programmable feeding and illumination. All functions are set by a user-friendly front panel for independent cycle programming. Bench testing with a model simulating the CO2 production of mice in the cage showed: a) suitable air renewal (by measuring actual ambient CO2), b) controlled realistic illumination at the mouse enclosure (measured by a photometer), c) stable temperature control, and d) correct cycling of light, feeding, and temperature. The cost of all the supplies (retail purchased by e-commerce) was <300 US$. Detailed technical information is open-source provided, allowing for any user to reliably reproduce or modify the device. This approach can considerably facilitate circadian research since using one of the described low-cost devices for any mouse group with a given light-food-temperature paradigm allows for all the experiments to be performed simultaneously, thereby requiring no changes in the light/temperature of a general-use laboratory. Copyright © 2024 Farré, Rodríguez-Lázaro, Otero, Gavara, Sunyer, Farré, Gozal and Almendros.

JTD Keywords: Animal experiment, Animal model, Animal research, Article, Circadian alteration, Circadian rhythm, Commercial phenomena, Controlled study, Cycling, Energy consumption, Experimental model, Feeding, Food, Food availability, Illumination, Intermittent fasting, Light, Light cycle, Light dark cycle, Mouse, Nonhuman, Open source technology, Open-source hardware, Performance, Photography, Research, Rodent, Temperature, Temperature cycle

Naso- and oropharyngeal swabs are the Center for Disease Control and Prevention (CDC) -recommended disease sampling methods for respiratory viruses. The short swabbing time for sampling by these methods may lead to variability in test results. Further, these methods are mildly invasive and can cause discomfort, tearing or gag reflexes in tested individuals. If longer sampling time is coupled with lesser patient discomfort, test reliability and patient compliance can be improved. Towards this end, we developed cationic biopolymer membranes for the electrostatic capturing of viruses in the oral cavity. Here, chemically (EDC-NHS) crosslinked uncharged chitosan (CS) nanofiber membranes were conferred either with negative surface charge by anionic poly-aspartic acid (pAsp) coating or positive charge by cationic poly-L-lysine (PLL). Consistent with our preliminary findings of dynamic light scattering (DLS) size measurements showing large agglomerates of anionic virus-like particles (VLPs) and cationic PLL in solution, a 75% increase in VLP adsorption by PLL coated CS membranes was recorded by enzyme linked immunosorbent assay (ELISA), in comparison to untreated controls. It is envisaged that the electrostatic concentration of respiratory viruses on cationic membranes can be superior alternatives to traditional swabbing in the oral cavity.

JTD Keywords: Cationic biopolymer membranes, Disease sampling, Dynamic light scattering (dls), Electrostatic capture of viruses, Enzyme linked immunosorbent assay (elisa), Magnetic beads, Virus -like particles (vlps)

Nanoparticles (NPs) are used to encapsulate therapeutic cargos and deliver them specifically to the target site. The intracellular trafficking of NPs dictates the NP-cargo distribution within different cellular compartments, and thus governs their efficacy and safety. Knowledge in this field is crucial to understand their biological fate and improve their rational design. However, there is a lack of methods that allow precise localization and quantification of individual NPs within distinct cellular compartments simultaneously. Here, we address this issue by proposing a correlative light and electron microscopy (CLEM) method combining direct stochastic optical reconstruction microscopy (dSTORM) and transmission electron microscopy (TEM). We aim at combining the advantages of both techniques to precisely address NP localization in the context of the cell ultrastructure. Individual fluorescently-labelled poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) NPs were directly visualized by dSTORM and assigned to cellular compartments by TEM. We first tracked NPs along the endo-lysosomal pathway at different time points, then demonstrated the effect of chloroquine on their intracellular distribution (i.e. endosomal escape). The proposed protocol can be applied to fluorescently labelled NPs and/or cargo, including those not detectable by TEM alone. Our studies are of great relevance to obtain important information on NP trafficking, and crucial for the design of more complex nanomaterials aimed at cytoplasmic/nucleic drug delivery.

JTD Keywords: chemistry, delivery, endocytosis, endosomal escape, exocytosis, fluorescence, light, size, tomography, Cellular uptake

Live cell actin visualization is fundamental for exploring cellular motility, cytokinesis, intracellular transport, and other correlated functions. The current imaging techniques that allow imaging of actin in its native environment are optical and electron microscopy. Such imaging techniques offer high enough resolution to investigate the ultrastructure of actin however they come at the expense of actin integrity. Inspired by the lack of suitable probes that preserve actin's integrity, we designed a cyclometalated Ir (III) complex that interacts with live cells and displays light switch behaviour upon specific actin binding. The exceptional photophysical properties of the proposed probe allow unprecedented resolution of cytoskeleton ultrastructures under stimulated emission depletion (STED) super-resolution nanoscopy. Moreover, the Ir complex enables the capability of visualizing actin polymers and periodicity under correlative light electron microscopy (CLEM) and liquid-phase electron microscopy (LPEM) at similar to 8 nm resolution.

JTD Keywords: Actin dynamics, Actin targeting, Adhesion, Cells, Clem, Fluorescent, Iridium (iii) complex, Lead, Light, Lpem, Super-resolution ultrastructures

The past ten years have seen the rapid expansion of the field of biohybrid robotics. By combining engineered, synthetic components with living biological materials, new robotics solutions have been developed that harness the adaptability of living muscles, the sensitivity of living sensory cells, and even the computational abilities of living neurons. Biohybrid robotics has taken the popular and scientific media by storm with advances in the field, moving biohybrid robotics out of science fiction and into real science and engineering. So how did we get here, and where should the field of biohybrid robotics go next? In this perspective, we first provide the historical context of crucial subareas of biohybrid robotics by reviewing the past 10+ years of advances in microorganism-bots and sperm-bots, cyborgs, and tissue-based robots. We then present critical challenges facing the field and provide our perspectives on the vital future steps toward creating autonomous living machines.

JTD Keywords: biohybrid, cyborg, Biohybrid, Cell, Cyborg, Delivery, Fabrication, Flight, Insect, Living machines, Muscle activities, Muscular thin-films, Nanoparticles, Stimulation, Tissue

Although an increasing number of micro/nanomotors have been designed for environmental remediation in the past decade, the construction of contaminants-fueled nanomotors for synergistically degrading multiple pollutants simultaneously remains a challenge. Herein, laccase-powered Fe3O4@silica nanomotors are fabricated, assisted with lipase enzyme for the enhanced degradation of multiple contaminants using the contaminants themselves as fuels. Notably, we demonstrate that representative industrial phenols and polycyclic aromatic pollutants possess the ability of triggering the enhanced Brownian motion of laccase nanomotors (De of 1.16 mu m(2)/s in 220 mu M biphenol A (BPA), 1.40 mu m(2)/s in 375 mu M Congo red (CR)). Additionally, the k(cat) value of lipase-assisted laccase-powered nanomotors increased over 1.4 times, enhancing their Brownian motion, while leading to the efficient degradation of multiple contaminants such as BPA, CR, and triacetin droplets within 40 min, simultaneously. Ultimately, the lipase-assisted laccase nanomotors exhibit great advantages over free laccase, free lipase, lipase nanomotors, or laccase nanomotors in K-m, k(cat), catalytic stability, recycling property, and the degradation efficiency of contaminants. Therefore, our work further broadens the library of enzyme-powered nanomotors and provides deep insights in synergistical enzymatic catalysis, thus paving avenues for environmental remediation based on enzyme-powered micro/nanomotors. (C) 2022 Elsevier Ltd. All rights reserved.

JTD Keywords: core, dye, environmental remediation, enzyme catalysis, hybrid, light, microspheres, motors, pollutants removal, propulsion, removal, self-propulsion, shell, Core, Dye, Environmental remediation, Enzyme catalysis, Hybrid, Light, Micro/nanomotors, Micromotors, Microspheres, Motors, Pollutants removal, Propulsion, Removal, Self-propulsion, Shell

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.

JTD Keywords: 3d architectures, biomolecular gradients, colon, crypt, engineering organoids, hydrogels, identification, in silico modeling, intestinal stem cell niches, light sheet fluorescence microscopy, niche, permeability, photolithography, regeneration, villus, wnt, 3d architectures, Biomolecular gradients, Engineering organoids, In silico modeling, Intestinal stem cell niches, Light sheet fluorescence microscopy, Photolithography, Stem-cell

The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues hinders the progress of the field. Here we report the development of OptoShroom3, an optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination in an epithelial Madin-Darby Canine Kidney (MDCK) cell sheet reduces the apical surface of the stimulated cells and causes displacements in the adjacent regions. Light-induced apical constriction provokes the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids leads to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.© 2022. The Author(s).

JTD Keywords: build, developmental biology, disease, light, localization, multicellular structures, organization, plate, shroom, Epithelial-cell shape

Photosynthesis is a fundamental process that converts photons into chemical energy, driven by large protein complexes at the thylakoid membranes of plants, cyanobacteria, and algae. In plants, water-soluble plastocyanin (Pc) is responsible for shuttling electrons between cytochrome b6f complex and the photosystem I (PSI) complex in the photosynthetic electron transport chain (PETC). For an efficient turnover, a transient complex must form between PSI and Pc in the PETC, which implies a balance between specificity and binding strength. Here, we studied the binding frequency and the unbinding force between suitably oriented plant PSI and Pc under redox control using single molecule force spectroscopy (SMFS). The binding frequency (observation of binding-unbinding events) between PSI and Pc depends on their respective redox states. The interaction between PSI and Pc is independent of the redox state of PSI when Pc is reduced, and it is disfavored in the dark (reduced P700) when Pc is oxidized. The frequency of interaction between PSI and Pc is higher when at least one of the partners is in a redox state ready for electron transfer (ET), and the post-ET situation (PSIRed-PcOx) leads to lower binding. In addition, we show that the binding of ET-ready PcRed to PSI can be regulated externally by Mg2+ ions in solution.

JTD Keywords: architecture, binding-site, complexes, ferredoxin, force spectroscopy, induced structural-changes, interprotein electron transfer, light-dependent interaction, mg2+ concentration, photosystem i, plastocyanin, probe, recognition, reduction, Force spectroscopy, Interprotein electron transfer, Light-dependent interaction, Photosynthetic reaction-center, Photosystem i, Plastocyanin, Single molecule measurements

The interest in the photochromism and functional applications of donor-acceptor Stenhouse adducts (DASAs) soared in recent years owing to their outstanding advantages and flexible design. However, their low solubility and irreversible conversion in aqueous solutions hampered exploring DASAs for biology and medicine. It is notably unknown whether the barbiturate electron acceptor group retains the pharmacological activity of drugs such as phenobarbital, which targets γ-aminobutyric acid (GABA)-type A receptors (GABAARs) in the brain. Here, we have developed the model compound DASA-barbital based on a scaffold of red-switching second-generation DASAs, and we demonstrate that it is active in GABAARs and alters the neuronal firing rate in a physiological medium at neutral pH. DASA-barbital can also be reversibly photoswitched in acidic aqueous solutions using cyclodextrin, an approved ingredient of drug formulations. These findings clarify the path toward the biological applications of DASAs and to exploit the versatility displayed in polymers and materials science.

JTD Keywords: behavior, receptor, visible-light, wavelength, Optical control

Over the last decades, photopharmacology has gone far beyond its proof-of-concept stage to become a bona fide approach to study neural systems in vivo. Indeed, photopharmacological control has expanded over a wide range of endogenous targets, such as receptors, ion channels, transporters, kinases, lipids, and DNA transcription processes. In this review, we provide an overview of the recent progresses in the in vivo photopharmacological control of neuronal circuits and behavior. In particular, the use of small aquatic animals for the in vivo screening of photopharmacological compounds, the recent advances in optical modulation of complex behaviors in mice, and the development of adjacent techniques for light and drug delivery in vivo are described.

JTD Keywords: brain circuits, circadian rhythm, in vivo photomodulation, in vivo technology, neuronal receptors, Architecture, Azobenzene photoswitches, Brain circuits, Channels, Circadian rhythm, In vivo photomodulation, In vivo technology, Light, Modulator, Neuronal receptors, Optical control, Optogenetics, Pharmacology, Photopharmacology, Receptors, Systems

The present work focuses on the computational study of the structural micro-organization of hydrogels based on collagen-like peptides (CLPs) in complex with Rose Bengal (RB). In previous studies, these hydrogels computationally and experimentally demonstrated that when RB was activated by green light, it could generate forms of stable crosslinked structures capable of regenerating biological tissues such as the skin and cornea. Here, we focus on the structural and atomic interactions of two collagen-like peptides (collagen-like peptide I (CLPI), and collagen-like peptide II, (CLPII)) in the presence and absence of RB, highlighting the acquired three-dimensional organization and going deep into the stabilization effect caused by the dye. Our results suggest that the dye could generate a ternary ground-state complex between collagen-like peptide fibers, specifically with positively charged amino acids (Lys in CLPI and Arg in CLPII), thus stabilizing ordered three-dimensional structures. The discoveries generated in this study provide the structural and atomic bases for the subsequent rational development of new synthetic peptides with improved characteristics for applications in the regeneration of biological tissues during photochemical tissue bonding therapies.

JTD Keywords: collagen-like peptide, crosslinking, molecular dynamics, qm/mm simulations, rose bengal, Anastomosis, Collagen-like peptide, Crosslinking, Green light, Mm simulations, Molecular dynamics, Molecular-dynamics, Photochemical tissue bonding therapies, Qm, Rose bengal

Three-dimensional bioprinting (3D bioprinting) has been at theforefront of tissue engineering research in the past years, with evermore efficient systems reaching the market(1). While existing 3Dbioprinting techniques are numerous and varied, they are limited bylong printing times when used at high resolution(2). The techniquedescribed in this work aims at enabling fast and accurate productionof monolayered skin constructs.To achieve shorter production times, a digital scanned light sheetis used to produce patterns of polymerized hydrogel, which enablesthe printing of a full three-dimensional plane in a matter of a fewhundred milliseconds. The high resolution resides in the properties ofthe light sheet itself – the width of the light sheet represents the z-axial resolution of the system (as low as 10mm) and the x-axialresolution is determined by the intensity profile of the gaussian beam(around 50mm). In order to fully exploit this system, the hydrogelused to encapsulate the cells must therefore be tailor-made for pho-toactivated cross-linking.As a proof of concept, a light sheet microscope is used as a po-lymerization source for novel photosensitive hydrogels. The up-coming hardware, software, chemical and biological improvementsneeded to reach the full potential of this system are expected toeventually be sufficient to print a complete skin construct, whichcould be used in the drug development industry, or as a graft forregenerative medicine therapy. Additionally, the constructs can beused to reduce and even replace animal testing for drug or cosmetictesting.

JTD Keywords: 3d bioprinting, Light sheet microscopy, Stereolithography

The endosomal sorting complex required for transport (ESCRT) machinery mediates membrane fission reactions that exhibit a different topology from that observed in clathrin-coated vesicles. In all of the ESCRT-mediated events, the nascent vesicle buds away from the cytosol. However, ESCRT proteins are able to act upon membranes with different geometries. For instance, the formation of multivesicular bodies (MVBs) and the biogenesis of extracellular vesicles both require the participation of the ESCRT-III sub-complex, and they differ in their initial membrane geometry before budding starts: the protein complex acts either from outside the membrane organelle (causing inward budding) or from within (causing outward budding). Several studies have reconstituted the action of the ESCRT-III subunits in supported bilayers and cell-sized vesicles mimicking the geometry occurring during MVBs formation (in-bud), but extracellular vesicle budding (out-bud) mechanisms remain less explored, because of the outstanding difficulties encountered in encapsulation of functional ESCRT-III in vesicles. Here, we provide a different approach that allows the recreation of the out-bud formation, by combining giant unilamellar vesicles as a membrane model and a microinjection system. The vesicles are immobilized prior to injection via weak adhesion to the chamber coverslip, which also ensures preserving the membrane excess area required for budding. After protein injection, vesicles exhibit outward budding. The approach presented in this work can be used in the future to disentangle the mechanisms underlying ESCRT-III-mediated fission, recreating the geometry of extracellular bud production, which remains a challenge. Moreover, the microinjection methodology can be also adapted to interrogate the action of other cytosolic components on the encapsulating membranous organelle. Copyright: © 2022 The Authors.

JTD Keywords: adhesion, budding, electroformation, escrt-iii, exosomes, extracellular vesicles, giant unilamellar vesicle (guv), light, microinjection, microparticles, plasma, Adhesion, Budding, Escrt-iii, Extracellular vesicles, Giant unilamellar vesicle (guv), Membrane, Microinjection

The identification of key components relevant to sensory perception of quality from commercial chocolate samples was accomplished after chemometric processing of GC×GC-MS (Comprehensive Two-dimensional Gas Chromatography with Mass Spectrometric Detection) profiles corresponding to HS-SPME (Headspace Solid Phase Microextraction) extracts of the samples. Descriptive sensory evaluation of samples was carried out using Optimized Descriptive Profile (ODP) procedures, where sensory attributes of 24 commercial chocolate samples were used to classify them in two classes (low and high chocolate flavor). 2D Fisher Ratio analysis was applied to four-way chromatographic data tensors (1st dimension retention time 1tR × 2nd dimension retention time 2tR × m/z × sample), to identify the crucial areas on the chromatograms that resulted on ODP class separation on Principal Component Analysis (PCA) scores plot. Comparing the relevant sections of the chromatograms to the analysis of the corresponding mass spectra, it was possible to assess that most of the information regarding the sample main sensory attributes can be related to only 14 compounds (2,5-dimethylpyrazine, 2,6-dimethyl-4-heptanol, 1-octen-3-ol, trimethylpyrazine, β-pinene, o-cimene, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, benzaldehyde, 1,3,5-trimethylbenzene, 6-methyl-5-hepten-2-one, limonene, benzeneethanol and 1,1-dimethylbutylbenzene) among the complex blend of volatiles found on these extremely complex samples.

JTD Keywords: classification, cocoa, dark chocolate, feature-selection, fisher ratio, gcxgc-ms, impact, olfactometry, principal component analysis, sensorial analysis, Chocolate flavor, Fisher ratio, Flight mass-spectrometry, Gc×gc-ms, Principal component analysis, Sensorial analysis

The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations-as in slow wave sleep-is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans.

JTD Keywords: brain states, light-mediated control, muscarinic acetylcholine receptors, neuromodulation, Activation, Alternating/direct currents, Basal forebrain, Brain, Brain states, Clinical research, Clinical translation, Controlled drug delivery, Cortex, Forebrain cholinergic system, Genetic manipulations, Higher frequencies, Hz oscillation, Light‐, Light-mediated control, Mediated control, Muscarinic acetylcholine receptors, Muscarinic agonists, Muscarinic receptor, Neurology, Neuromodulation, Neurons, Noradrenergic modulation, Parvalbumin-positive interneurons, Photopharmacology, Receptor-binding, Slow, Spatiotemporal control, Spatiotemporal patterns

A new methodology for the pH-triggered degradation of polymers or for the release of drugs under visible light irradiation based on the cyclization of ortho-hydroxy-cinnamates (oHC) to coumarins is described. The key oHC structural motif can be readily incorporated into the rational design of novel photocleavable polymers via click chemistry. This main-chain moiety undergoes a fast photocleavage when irradiated with 455 nm light provided that a suitable base is added. A series of polyethylene glycol-alt-ortho-hydroxy cinnamate (polyethylene glycol (PEG)(n)-alt-oHC)-based polymers are synthesized and the time-dependent visible-light initiated cleavage of the photoactive monomer and polymer is investigated in solution by a variety of spectroscopic and chromatographic techniques. The photo-degradation behavior of the water-soluble poly(PEG(2000)-alt-oHC) is investigated within a broad pH range (pH = 2.1-11.8), demonstrating fast degradation at pH 11.8, while the stability of the polymer is greatly enhanced at pH 2.1. Moreover, the neat polymer shows long-term stability under daylight conditions, thus allowing its storage without special precautions. In addition, two water-soluble PEG-based drug-carrier molecules (mPEG(2000)-oHC-benzhydrol/phenol) are synthesized and used for drug delivery studies, monitoring the process by UV-vis spectroscopy in an ON/OFF intermittent manner.

JTD Keywords: coumarins, drug delivery, e/z-double bond isomerization, o-hydroxy cinnamates, polymer degradation, Aliphatic compounds, Antioxidant activity, Antitumor, Chromatographic techniques, Chromatography, Cis/trans isomerization, Controlled drug delivery, Coumarin derivatives, Coumarins, Drug delivery, Drug delivery system, E/z-double bond isomerization, Films, Hydrogels, Image enhancement, Light, Long term stability, O-hydroxy cinnamates, Particles, Photoactive monomers, Photodegradation, Polyethylene glycols, Polyethylenes, Polymer degradation, Responsive polymers, Salts, Structural motifs, Synthesis (chemical), Targeted drug delivery, Visible light photocatalysis, Visible-light irradiation

The functionalization of nanoparticles with functional moieties is a key strategy to achieve cell targeting in nanomedicine. The interplay between size and ligand number is crucial for the formulation performance and needs to be properly characterized to understand nanoparticle structure-activity relations. However, there is a lack of methods able to measure both size and ligand number at the same time and at the single particle level. Here, we address this issue by introducing a correlative light and electron microscopy (CLEM) method combining super-resolution microscopy (SRM) and transmission electron microscopy (TEM) imaging. We apply our super-resCLEM method to characterize the relationship between size and ligand number and density in PLGA-PEG nanoparticles. We highlight how heterogeneity found in size can impact ligand distribution and how a significant part of the nanoparticle population goes completely undetected in the single-technique analysis. Super-resCLEM holds great promise for the multiparametric analysis of other parameters and nanomaterials.

JTD Keywords: cellular uptake, correlative light and electron microscopy (clem), density, electron microscopy (em), functionalization, heterogeneity, nanomedicine, nanoparticles, pegylation, plga, progress, quantification, size, Correlative light and electron microscopy (clem), Electron microscopy (em), Heterogeneity, Nanomedicine, Nanoparticles, Physicochemical characterization, Super-resolution microscopy (srm)

© 2020 Elsevier Inc. Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples.

JTD Keywords: cells, click-chemistry, complex, correlative light and electron microscopy, cycloaddition, ligation, localization, proteins, resolution limit, single molecule localization microscopy, stochastic optical reconstruction microscopy (storm), storm, super-resolution microscopy, tokuyasu cryo-sectioning, tool, Click-chemistry, Correlative light and electron microscopy, Fluorescent-probes, Single molecule localization microscopy, Stochastic optical reconstruction microscopy (storm), Super-resolution microscopy, Tokuyasu cryo-sectioning, Transmission electron microscopy

The visualization of microtubules by combining optical and electron microscopy techniques provides valuable information to understand correlated intracellular activities. However, the lack of appropriate probes to bridge both microscopic resolutions restricts the areas and structures that can be comprehended within such highly assembled structures. Here, a versatile cyclometalated iridium (III) complex is designed that achieves synchronous fluorescence–electron microscopy correlation. The selective insertion of the probe into a microtubule triggers remarkable fluorescence enhancement and promising electron contrast. The long-life, highly photostable probe allows live-cell super-resolution imaging of tubulin localization and motion with a resolution of ≈30 nm. Furthermore, correlative light–electron microscopy and energy-filtered transmission electron microscopy reveal the well-associated optical and electron signal at a high specificity, with an interspace of ≈41 Å of microtubule monomer in cells.

JTD Keywords: Correlation light–electron microscopy, Microtubules, Organometallic probes, Super-resolution microscopy

The motion of nanomotors triggered by light sources will provide new alternative routes to power nanoarchitectures without the need of chemical fuels. However, most light-driven nanomotors are triggered by UV-light, near infrared reflection, or laser sources. It is demonstrated that nanocap shaped Au/TiO2 nanomotors (175 nm in diameter) display increased Brownian motion in the presence of broad spectrum visible light. The motion results from the surface plasmon resonance effect leading to self-electrophoresis between the Au and TiO2 layers, a mechanism called plasmonic photocatalytic effect in the field of photocatalysis. This mechanism is experimentally characterized by electron energy loss spectroscopy, energy-filtered transmission electron microscopy, and optical video tracking. This mechanism is also studied in a more theoretical manner using numerical finite-difference time-domain simulations. The ability to power nanomaterials with visible light may result in entirely new applications for externally powered micro/nanomotors.

JTD Keywords: Enhanced Brownian motion, Fuel-free nanomotors, Nanomachines, Self-electrophoresis, Visible light

Background Neurons signal to each other and to non-neuronal cells as those in muscle or glands, by means of the secretion of neurotransmitters at chemical synapses. In order to dissect the molecular mechanisms of neurotransmission, new methods for directly and reversibly triggering neurosecretion at the presynaptic terminal are necessary. Here we exploit the calcium permeability of the light-gated channel LiGluR in order to reversibly manipulate cytosolic calcium concentration, thus controlling calcium-regulated exocytosis. Methods Bovine chromaffin cells expressing LiGluR were stimulated with light. Exocytic events were detected by amperometry or by whole-cell patch-clamp to quantify membrane capacitance and calcium influx. Results Amperometry reveals that optical stimulation consistently triggers exocytosis in chromaffin cells. Secretion of catecholamines can be adjusted between zero and several Hz by changing the wavelength of illumination. Differences in secretion efficacy are found between the activation of LiGluR and native voltage-gated calcium channels (VGCCs). Our results show that the distance between sites of calcium influx and vesicles ready to be released is longer when calcium influx is triggered by LiGluR instead of native VGCCs. Conclusion and general significance LiGluR activation directly and reversibly increases the intracellular calcium concentration. Light-gated calcium influx allows for the first time to control calcium-regulated exocytosis without the need of applying depolarizing solutions or voltage clamping in chromaffin cells. Thus, LiGluR is a useful tool to study the secretory mechanisms and their spatiotemporal patterns in neurotransmission, and opens a window to study other calcium-dependent processes such as muscular contraction or cell migration.

JTD Keywords: Optical control, Calcium, Exocytosis, Light-gated glutamate receptor (LiGluR), Neurotransmission, Optogenetics

Although the identity and interactions of signaling proteins have been studied in great detail, the complexity of signaling networks cannot be fully understood without elucidating the timing and location of activity of individual proteins. To do this, one needs a means for detecting and controlling specific signaling events. An attractive approach is to use light, both to report on and control signaling proteins in cells, because light can probe cells in real time with minimal damage. Although optical detection of signaling events has been successful for some time, the development of the means for optical control has accelerated only recently. Of particular interest is the development of chemically engineered proteins that are directly sensitive to light.

JTD Keywords: Ion channels, Acetylcholine receptor, Glutamate-receptor, Potassium channel, K+ channel, Light, Neurons, Channelrhodopsin-2, Manipulation, Activation