Publications

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.

JTD Keywords: Ai, Consumption, Culture, Embryo development, Fluorescence, Hyperspectral imagin, Implantation, In vitro fertilization, Infertility, Label-free imaging, Microscopy, Morphokinetics, Oxygen concentrations, Selectio, Time-lapse

Viability and vitality assays play a crucial role in assessing the effectiveness of novel therapeutic approaches, with stain-based methods providing speed and objectivity. However, their application in yeast research lacks consensus. This study aimed to assess the performance of four common dyes on C. parapsilosis planktonic cells as well as sessile cells that form well-structured biofilms (treated and not treated with amphotericin B). Viability assessment employed Syto-9 (S9), thiazole orange (TO), and propidium iodide (PI). Metabolic activity was determined using fluorescein diacetate (FDA) and FUN-1. Calcofluor white (CW) served as the cell visualization control. Viability/vitality percentage of treated samples were calculated for each dye from confocal images and compared to crystal violet and PrestoBlue results. Heterogeneity in fluorescence intensity and permeability issues were observed with S9, TO, and FDA in planktonic cells and biofilms. This variability, influenced by cell morphology, resulted in dye-dependent viability/vitality percentages. Notably, PI and FUN-1 exhibited robust C. parapsilosis staining, with FUN-1 vitality results comparable to PrestoBlue. Our finding emphasizes the importance of evaluating dye permeability in yeast species beforehand, incorporating cell visualization controls. An improper dye selection may lead to misinterpreting treatment efficacy.

JTD Keywords: Albicans,quantification,biomass,image,aci, Biofilms,microscopy,imaging,amphotericin b,stain-based methods,yeast staining,fluorescence,live and dea

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

JTD Keywords: Biofabrication,full-thickness skin model,light sheet bioprinter,light sheet fluorescence microscopy,tissue engineerin

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, Chemistry, Delivery, Endocytosis, Endosomal escape, Exocytosis, Fluorescence, Light, Size, Tomography

Ratiometric fluorescent nanothermometers with near-infrared emission play an important role in in vivo sensing since they can be used as intracellular thermal sensing probes with high spatial resolution and high sensitivity, to investigate cellular functions of interest in diagnosis and therapy, where current approaches are not effective. Herein, the temperature-dependent fluorescence of organic nanoparticles is designed, synthesized, and studied based on the dual emission, generated by monomer and excimer species, of the tris(2,4,6-trichlorophenyl)methyl radical (TTM) doping organic nanoparticles (TTMd-ONPs), made of optically neutral tris(2,4,6-trichlorophenyl)methane (TTM-αH), acting as a matrix. The excimer emission intensity of TTMd-ONPs decreases with increasing temperatures whereas the monomer emission is almost independent and can be used as an internal reference. TTMd-ONPs show a great temperature sensitivity (3.4% K-1 at 328 K) and a wide temperature response at ambient conditions with excellent reversibility and high colloidal stability. In addition, TTMd-ONPs are not cytotoxic and their ratiometric outputs are unaffected by changes in the environment. Individual TTMd-ONPs are able to sense temperature changes at the nano-microscale. In vivo thermometry experiments in Caenorhabditis elegans (C. elegans) worms show that TTMd-ONPs can locally monitor internal body temperature changes with spatio-temporal resolution and high sensitivity, offering multiple applications in the biological nanothermometry field.© 2023 The Authors. Small published by Wiley-VCH GmbH.

JTD Keywords: dual emission, elegans, excimer emission, fluorescence, in vivo sensing, luminescence, nanoparticles, organic radical nanoparticles, ratiometric nanothermometers, sensors, thermometry, trityl radicals, Caenorhabditis elegans, Excimer emission, In vivo sensing, Intracellular ph, Luminescence, Organic radical nanoparticles, Ratiometric nanothermometers, Trityl radicals

Supramolecular systems chemistry has been an area of active research to develop nanomaterials with life-like functions. Progress in systems chemistry relies on our ability to probe the nanostructure formation in solution. Often visualizing the dynamics of nanostructures which transform over time is a formidable challenge. This necessitates a paradigm shift from dry sample imaging towards solution-based techniques. We review the application of state-of-the-art techniques for real-time, in situ visualization of dynamic self-assembly processes. We present how solution-based techniques namely optical super-resolution microscopy, solution-state atomic force microscopy, liquid-phase transmission electron microscopy, molecular dynamics simulations and other emerging techniques are revolutionizing our understanding of active and adaptive nanomaterials with life-like functions. This Review provides the visualization toolbox and futuristic vision to tap the potential of dynamic nanomaterials.© 2022 Wiley-VCH GmbH.

JTD Keywords: electron-microscopy, fluorescence microscopy, in-situ, mechanical-properties, molecular simulations, nanostructures, polymerization, polymers, stimulated-emission, super-resolution microscopy, supramolecular chemistry, systems chemistry, water, Atomic-force microscopy, Liquid tem, Nanostructures, Super-resolution microscopy, Supramolecular chemistry, Systems chemistry

Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.© 2022. The Author(s), under exclusive licence to Springer Nature Limited.

JTD Keywords: colonization, defines, human colon, mutations, plasticity, retrieval, stem-cells, subtypes, underlie, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Article, Cancer, Cancer growth, Cancer immunotherapy, Cancer inhibition, Cancer recurrence, Cancer staging, Cell, Cell adhesion, Cell migration, Cell population, Cell surface receptor, Cohort analysis, Colorectal cancer, Colorectal neoplasms, Colorectal tumor, Comprehensive molecular characterization, Controlled study, Crispr-cas9 system, Cytoskeleton, Disease exacerbation, Disease progression, Dynamics, Emp1 gene, Epithelial membrane protein-1, Extracellular matrix, Flow cytometry, Fluorescence intensity, Gene expression, Genetics, Human, Human cell, Humans, Immune response, Immunofluorescence, In situ hybridization, Marker gene, Metastasis potential, Mice, Minimal residual disease, Mouse, Neoplasm proteins, Neoplasm recurrence, local, Neoplasm, residual, Nonhuman, Pathology, Phenotype, Prevention and control, Protein, Receptors, cell surface, Single cell rna seq, Tumor, Tumor protein, Tumor recurrence

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

Nanoparticles represent a promising class of material for nanomedicine and molecular biosensing. The formation of a protein corona due to nonspecific particle-protein interactions is a determining factor for the biological fate of nanoparticles in vivo and strongly impacts the performance of nanoparticles when used as biosensors. Nonspecific interactions are usually highly heterogeneous, yet little is known about the heterogeneity of the protein corona that may lead to inter- and intraparticle differences in composition and protein distribution. Here, we present a super-resolution microscopic approach to study the protein corona on single silica nanoparticles and subsequent cellular interactions using multicolor stimulated emission depletion (STED) microscopy. We demonstrate that STED resolves structural features of protein corona on single particles including the distribution on the particle surface and the degree of protein internalization in porous particles. Using multicolor measurements of multiple labeled protein species, we determine the composition of the protein corona at the single-particle level. We quantify particle-to-particle differences in the composition and find that the composition is considerably influenced by the particle geometry. In a subsequent cellular uptake measurement, we demonstrate multicolor STED of protein corona on single particles internalized by cells. Our study shows that STED microscopy opens the window toward mechanistic understanding of protein coronas and aids in the rational design of nanoparticles as nanomedicines and biosensors.

JTD Keywords: insights, multicolor microscopy, nanoparticles, protein corona, quantification, size, sted microscopy, Fluorescence, Quantification, Sted microscopy

Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).Published by Elsevier B.V.

JTD Keywords: cell fusion, expression, fusion, ganglion-cells, in-vitro, mouse, müller glia, neural differentiation, organoids, regeneration, retina regeneration, stem cells, stromal cells, transplantation, 4',6 diamidino 2 phenylindole, 5' nucleotidase, Agarose, Alcohol, Arpe-19 cell line, Article, Beta catenin, Beta tubulin, Bone-marrow-cells, Bromophenol blue, Buffer, Calcium cell level, Calcium phosphate, Calretinin, Canonical wnt signaling, Cd34 antigen, Cell culture, Cell fusion, Cell viability, Coculture, Complementary dna, Confocal microscopy, Cornea transplantation, Cryopreservation, Cryoprotection, Crystal structure, Current clamp technique, Dimethyl sulfoxide, Dodecyl sulfate sodium, Edetic acid, Electrophysiology, Endoglin, Fetal bovine serum, Fibroblast growth factor 2, Flow cytometry, Fluorescence activated cell sorting, Fluorescence intensity, Glyceraldehyde 3 phosphate dehydrogenase, Glycerol, Glycine, Hoe 33342, Immunofluorescence, Immunohistochemistry, Incubation time, Interleukin 1beta, Lentivirus vector, Matrigel, Mercaptoethanol, Microinjection, Mueller cell, Müller glia, N methyl dextro aspartic acid, Nerve cell differentiation, Neural differentiation, Nitrogen, Nonhuman, Organoids, Paraffin, Paraffin embedding, Paraformaldehyde, Patch clamp technique, Penicillin derivative, Phenolsulfonphthalein, Phenotype, Phosphate buffered saline, Phosphoprotein phosphatase inhibitor, Polyacrylamide gel electrophoresis, Potassium chloride, Povidone iodine, Promoter region, Proteinase inhibitor, Real time polymerase chain reaction, Receptor type tyrosine protein phosphatase c, Restriction endonuclease, Retina, Retina dystrophy, Retina regeneration, Retinol, Rhodopsin, Rna extraction, Stem cell, Stem cells, Subcutaneous fat, Tunel assay, Visual impairment, Western blotting

The characterization of newly synthesized materials is a cornerstone of all chemistry and nanotechnology laboratories. For this purpose, a wide array of analytical techniques have been standardized and are used routinely by laboratories across the globe. With these methods we can understand the structure, dynamics and function of novel molecular architectures and their relations with the desired performance, guiding the development of the next generation of materials. Moreover, one of the challenges in materials chemistry is the lack of reproducibility due to improper publishing of the sample preparation protocol. In this context, the recent adoption of the reporting standard MIRIBEL (Minimum Information Reporting in Bio–Nano Experimental Literature) for material characterization and details of experimental protocols aims to provide complete, reproducible and reliable sample preparation for the scientific community. Thus, MIRIBEL should be immediately adopted in publications by scientific journals to overcome this challenge. Besides current standard spectroscopy and microscopy techniques, there is a constant development of novel technologies that aim to help chemists unveil the structure of complex materials. Among them super-resolution microscopy (SRM), an optical technique that bypasses the diffraction limit of light, has facilitated the study of synthetic materials with multicolor ability and minimal invasiveness at nanometric resolution. Although still in its infancy, the potential of SRM to unveil the structure, dynamics and function of complex synthetic architectures has been highlighted in pioneering reports during the last few years. Currently, SRM is a sophisticated technique with many challenges in sample preparation, data analysis, environmental control and automation, and moreover the instrumentation is still expensive. Therefore, SRM is currently limited to expert users and is not implemented in characterization routines. This perspective discusses the potential of SRM to transition from a niche technique to a standard routine method for material characterization. We propose a roadmap for the necessary developments required for this purpose based on a collaborative effort from scientists and engineers across disciplines.

JTD Keywords: blinking, fluorophore, intramolecular spirocyclization, localization, nanoparticles, resolution limit, reveals, single-molecule fluorescence, stimulated-emission, Characterization techniques, Diffraction, Distributed computer systems, Environmental management, Information reporting, Material chemistry, Materials characterization, Minimum information, Optical reconstruction microscopy, Optical resolving power, Sample preparation, Structure dynamics, Structure functions, Super-resolution microscopy, Synthesized materials

Tissue decellularization is typically assessed through absorbance-based DNA quantification after tissue digestion. This method has several disadvantages, namely its destructive nature and inadequacy in experimental situations where tissue is scarce. Here, we present an image processing algorithm for quantitative analysis of DNA content in (de)cellularized tissues as a faster, simpler and more comprehensive alternative. Our method uses local entropy measurements of a phase contrast image to create a mask, which is then applied to corresponding nuclei labelled (UV) images to extract average fluorescence intensities as an estimate of DNA content. The method can be used on native or decellularized tissue to quantify DNA content, thus allowing quantitative assessment of decellularization procedures. We confirm that our new method yields results in line with those obtained using the standard DNA quantification method and that it is successful for both lung and heart tissues. We are also able to accurately obtain a timeline of decreasing DNA content with increased incubation time with a decellularizing agent. Finally, the identified masks can also be applied to additional fluorescence images of immunostained proteins such as collagen or elastin, thus allowing further image-based tissue characterization.

JTD Keywords: decellularization, differentiation, fluorescence image, image processing, microscopic image, Decellularization, Fluorescence image, Image processing, Matrix, Microscopic image, Segmentation

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, Ligands, Microscopy, electron, transmission, Microscopy, fluorescence, Nanomedicine, Nanoparticles, Physicochemical characterization, Super-resolution microscopy (srm)

© 2020, Springer Science+Business Media, LLC, part of Springer Nature. In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell–cell interactions and cell–matrix adhesions. Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.

JTD Keywords: 3d spheroid invasion, cancer associated fibroblasts, collective migration, dissemination, epithelial cancer cells, leader/follower cells, 3d spheroid invasion, Cancer associated fibroblasts, Cancer-associated fibroblasts, Cell culture techniques, Cell line, tumor, Cell movement, Cell tracking, Collective invasion, Collective migration, Epithelial cancer cells, Extracellular matrix, Humans, Imaging, three-dimensional, Leader/follower cells, Microscopy, fluorescence, Spheroids, cellular, Tumor cells, cultured

© 2021, The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature. Our understanding in the inherent properties of human pluripotent stem cells (hPSCs) have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids. Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensions to generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We also provide a concise description on how to assess renal commitment during the time course of kidney organoid generation. This includes the use of flow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.

JTD Keywords: 2d monolayer, 3d organotypic culture, differentiation, flow cytometry, human pluripotent stem cells, immunocytochemistry, intermediate mesoderm, kidney organoid, nephron progenitor cells, nephrons, primitive streak, 2d monolayer, 3d organotypic culture, Cell culture techniques, Cell differentiation, Cells, cultured, Differentiation, Flow cytometry, Fluorescent antibody technique, Gene expression regulation, developmental, Human pluripotent stem cells, Humans, Immunocytochemistry, Intermediate mesoderm, Kidney, Kidney organoid, Microscopy, fluorescence, Morphogenesis, Nephron progenitor cells, Nephrons, Organoids, Pluripotent stem cells, Primitive streak, Signal transduction, Time factors, Tissue, Tissue engineering

At high temperatures, isoprene-emitting plants display a higher photosynthetic rate and a lower nonphotochemical quenching (NPQ) compared with nonemitting plants. The mechanism of this phenomenon, which may be very important under current climate warming, is still elusive. NPQ was dissected into its components, and chlorophyll fluorescence lifetime imaging microscopy (FLIM) was used to analyse the dynamics of excited chlorophyll relaxation in isoprene-emitting and nonemitting plants. Thylakoid membrane stiffness was also measured using atomic force microscope (AFM) to identify a possible mode of action of isoprene in improving photochemical efficiency and photosynthetic stability. We show that, when compared with nonemitters, isoprene-emitting tobacco plants exposed at high temperatures display a reduced increase of the NPQ energy-dependent component (qE) and stable (1) chlorophyll fluorescence lifetime; (2) amplitude of the fluorescence decay components; and (3) thylakoid membrane stiffness. Our study shows for the first time that isoprene maintains PSII stability at high temperatures by preventing the modifications of the surrounding environment, namely providing a more steady and homogeneous distribution of the light-absorbing centres and a stable thylakoid membrane stiffness. Isoprene photoprotects leaves with a mechanism alternative to NPQ, enabling plants to maintain a high photosynthetic rate at rising temperatures.

JTD Keywords: (High) temperature, Atomic force microscopy (AFM), Chlorophyll fluorescence (quenching and lifetime), Fluorescence lifetime imaging microscopy (FLIM), Isoprene, Nonphotochemical quenching (NPQ), Photosynthesis

The mechanical properties of the extracellular matrix (ECM)–a complex, 3D, fibrillar scaffold of cells in physiological environments–modulate cell behavior and can drive tissue morphogenesis, regeneration, and disease progression. For simplicity, it is often convenient to assume these properties to be time-invariant. In living systems, however, cells dynamically remodel the ECM and create time-dependent local microenvironments. Here, we show how cell-generated contractile forces produce substantial irreversible changes to the density and architecture of physiologically relevant ECMs–collagen I and fibrin–in a matter of minutes. We measure the 3D deformation profiles of the ECM surrounding cancer and endothelial cells during stages when force generation is active or inactive. We further correlate these ECM measurements to both discrete fiber simulations that incorporate fiber crosslink unbinding kinetics and continuum-scale simulations that account for viscoplastic and damage features. Our findings further confirm that plasticity, as a mechanical law to capture remodeling in these networks, is fundamentally tied to material damage via force-driven unbinding of fiber crosslinks. These results characterize in a multiscale manner the dynamic nature of the mechanical environment of physiologically mimicking cell-in-gel systems.

JTD Keywords: Collagens, Fibrin, Extracellular matrix, Cross-linking, Cell physiology, Deformation, Fluorescence imaging, Cell biology

Recently, we disclosed primaquine cell penetrating peptide conjugates that were more potent than parent primaquine against liver stage Plasmodium parasites and non-toxic to hepatocytes. The same strategy was now applied to the blood-stage antimalarial chloroquine, using a wide set of peptides, including TP10, a cell penetrating peptide with intrinsic antiplasmodial activity. Chloroquine-TP10 conjugates displaying higher antiplasmodial activity than the parent TP10 peptide were identified, at the cost of an increased hemolytic activity, which was further confirmed for their primaquine analogues. Fluorescence microscopy and flow cytometry suggest that these drug-peptide conjugates strongly bind, and likely destroy, erythrocyte membranes. Taken together, the results herein reported put forward that coupling antimalarial aminoquinolines to cell penetrating peptides delivers hemolytic conjugates. Hence, despite their widely reported advantages as carriers for many different types of cargo, from small drugs to biomacromolecules, cell penetrating peptides seem unsuitable for safe intracellular delivery of antimalarial aminoquinolines due to hemolysis issues. This highlights the relevance of paying attention to hemolytic effects of cell penetrating peptide-drug conjugates.

JTD Keywords: Antimalarial, Cell penetrating peptide, Chloroquine, Erythrocyte fluorescence, Flow cytometry, Hemolysis, Microscopy, Plasmodium, Primaquine, Red blood cell

Synapses learn and remember by persistent modifications of their internal structures and composition but, due to their small size, it is difficult to observe these changes at the ultrastructural level in real time. Two-photon fluorescence microscopy (2PM) allows time-course live imaging of individual synapses but lacks ultrastructural resolution. Electron microscopy (EM) allows the ultrastructural imaging of subcellular components but cannot detect fluorescence and lacks temporal resolution. Here, we describe a combination of procedures designed to achieve the correlated imaging of the same individual synapse under both 2PM and EM. This technique permits the selective stimulation and live imaging of a single dendritic spine and the subsequent localization of the same spine in EM ultrathin serial sections. Landmarks created through a photomarking method based on the 2-photon-induced precipitation of an electrodense compound are used to unequivocally localize the stimulated synapse. This technique was developed to image, for the first time, the ultrastructure of the postsynaptic density in which long-term potentiation was selectively induced just seconds or minutes before, but it can be applied for the study of any biological process that requires the precise relocalization of micron-wide structures for their correlated imaging with 2PM and EM.

JTD Keywords: Correlated imaging, DAB, Dendritic spine, Photobranding, Photoetching, Photomarking, Postsynaptic density, Serial-section transmission electron microscopy, Synapse, Time-lapse live two-photon fluorescence microscopy

Heparin had been demonstrated to have antimalarial activity and specific binding affinity for Plasmodium-infected red blood cells (pRBCs) vs. non-infected erythrocytes. Here we have explored if both properties could be joined into a drug delivery strategy where heparin would have a dual role as antimalarial and as a targeting element of drug-loaded nanoparticles. Confocal fluorescence and transmission electron microscopy data show that after 30. min of being added to living pRBCs fluorescein-labeled heparin colocalizes with the intracellular parasites. Heparin electrostatically adsorbed onto positively charged liposomes containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane and loaded with the antimalarial drug primaquine was capable of increasing three-fold the activity of encapsulated drug in Plasmodium falciparum cultures. At concentrations below those inducing anticoagulation of mouse blood in vivo, parasiticidal activity was found to be the additive result of the separate activities of free heparin as antimalarial and of liposome-bound heparin as targeting element for encapsulated primaquine. From the Clinical Editor: Malaria remains an enormous global public health concern. In this study, a novel functionalized heparin formulation used as drug delivery agent for primaquine was demonstrated to result in threefold increased drug activity in cell cultures, and in a murine model it was able to provide these benefits in concentrations below what would be required for anticoagulation. Further studies are needed determine if this approach is applicable in the human disease as well.

JTD Keywords: Heparin, Liposomes, Malaria, Plasmodium, Targeted drug delivery, Heparin, Malaria, Plasmodium, Red blood cell, Targeted drug delivery, Liposomes, 1,2 dioleoyl 3 trimethylammoniopropane, fluorescein, heparin, liposome, nanoparticle, primaquine, adsorption, animal experiment, anticoagulation, antimalarial activity, Article, binding affinity, confocal microscopy, controlled study, drug targeting, encapsulation, erythrocyte, female, fluorescence microscopy, human, human cell, in vivo study, liposomal delivery, mouse, nonhuman, Plasmodium falciparum, transmission electron microscopy

Near-field optical microscopy is a technique not limited by the laws of diffraction that enables simultaneous high-resolution fluorescence and topographic measurements at the nanometer scale. This chapter highlights the intrinsic advantages of near-field optics in the study of cellular structures. The first part of the chapter lays the foundations of the near-field concept and technical implementation of near-field scanning optical microscopy (NSOM), whereas the second part of the chapter focuses on applications of NSOM to the study of model membranes and cellular structures on the plasma membrane. The last part of the chapter discusses further directions of near-field optics, including optical antennas and fluorescence correlation spectroscopy approaches in the near-field regime.

JTD Keywords: Biological membranes, Cell membrane nanoscale compartmentalization, Cellular nanodomains, Fluorescence correlation spectroscopy in reduced volumes, Immunoreceptor imaging, Lipid rafts, Near-field scanning optical microscopy, Optical nano-antennas, Shear force imaging, Single molecule detection, Super-resolution microscopy

We report a new strategy for immunochemical screening of small organic molecules based on the use of a hapten microarray. Using DNA-directed immobilization strategies, we have been able to convert a DNA chip into a hapten microarray by taking advantage of all the benefits of the structural and electrostatic homogeneous properties of DNA. The hapten microarray uses hapten-oligonucleotide probes instead of proteins, avoiding the limitations of preparing stochiometrically-defined protein-oligonucleotide bioconjugates. As proof of concept, we show here the development of a microarray for analysis of anabolic androgenic steroids. The microchip is able to detect several illegal substances with sufficient detectability to be used as a screening method, according to the regulations of the World Anti-Doping Agency for sport and the European Commision for food safety. The results that we show corroborate the universal possibilities of the DNA chip, and, in this case, they open the way to develop hapten microarrays for the immunochemical analysis of small organic molecules.

JTD Keywords: Anti-doping, DNA chip, DNA-directed immobilization (DDI), Fluorescence, Food safety, Hapten microarray, Immunochemical screening, Proof of concept, Small organic molecule, Steroid

In this work the presence of inverted hexagonal phases HII of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca2+ were observed via 31P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, HII phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being ∼ 1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to HII phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates (∼ 100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from HII phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.

JTD Keywords: 31P-NMR, AFM, ANS fluorescence, Cytochrome c (cyt c), Hexagonal phase (HII), Liposome, Supported planar bilayers (SPBs)