In dynamic light microscopy applications, imaging specimens from multiple angles while maintaining controlled temperature conditions is crucial for comprehensive and accurate analysis. To address these challenges, we present iCAT, an open-source multifunctional accessory designed to enhance light microscopy. It enables specimen rotation along the axial plane, incorporates built-in modules for precise temperature control, features an integrated LED, and includes a camera for real-time specimen monitoring. It can be easily 3D-printed and assembled using readily available electrical components. Combined with any up-right microscope, this versatile device allows researchers to capture detailed images and videos of organoid cultures and live or fixed specimens, such as C. elegans, zebrafish, drosophila, or mouse embryos. The potential applications of iCAT in investigating dynamic cellular processes and complex developmental phenomena are vast, inspiring researchers to explore its possibilities and push the boundaries of biological research.

Development of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from ventral aortic endothelium in all vertebrate animals studied. Work in zebrafish has demonstrated that arterial and venous endothelial precursors arise from distinct subsets of lateral plate mesoderm. Here, we profile the transcriptome of the earliest detectable endothelial cells (ECs) during zebrafish embryogenesis to demonstrate that tissue-specific EC programs initiate much earlier than previously appreciated, by the end of gastrulation. Classic studies in the chick embryo showed that paraxial mesoderm generates a subset of somite-derived endothelial cells (SDECs) that incorporate into the dorsal aorta to replace HSPCs as they exit the aorta and enter circulation. We describe a conserved program in the zebrafish, where a rare population of endothelial precursors delaminates from the dermomyotome to incorporate exclusively into the developing dorsal aorta. Although SDECs lack hematopoietic potential, they act as a local niche to support the emergence of HSPCs from neighboring hemogenic endothelium. Thus, at least three subsets of ECs contribute to the developing dorsal aorta: vascular ECs, hemogenic ECs, and SDECs. Taken together, our findings indicate that the distinct spatial origins of endothelial precursors dictate different cellular potentials within the developing dorsal aorta.

The cellular pathology of schizophrenia and the potential of antipsychotics to target underlying neuronal dysfunctions are still largely unknown. We employed glutamatergic neurons derived from induced pluripotent stem cells (iPSC) obtained from schizophrenia patients with known histories of response to clozapine and healthy controls to decipher the mechanisms of action of clozapine, spanning from molecular (transcriptomic profiling) and cellular (electrophysiology) levels to observed clinical effects in living patients. Glutamatergic neurons derived from schizophrenia patients exhibited deficits in intrinsic electrophysiological properties, synaptic function and network activity. Deficits in K+ and Na+ currents, network behavior, and glutamatergic synaptic signaling were restored by clozapine treatment, but only in neurons from clozapine-responsive patients. Moreover, neurons from clozapine-responsive patients exhibited a reciprocal dysregulation of gene expression, particularly related to glutamatergic and downstream signaling, which was reversed by clozapine treatment. Only neurons from clozapine responders showed return to normal function and transcriptomic profile. Our results underscore the importance of K+ and Na+ channels and glutamatergic synaptic signaling in the pathogenesis of schizophrenia and demonstrate that clozapine might act by normalizing perturbances in this signaling pathway. To our knowledge this is the first study to demonstrate that schizophrenia iPSC-derived neurons exhibit a response phenotype correlated with clinical response to an antipsychotic. This opens a new avenue in the search for an effective treatment agent tailored to the needs of individual patients.

Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. The CSF1R pathway is a promising therapeutic target in many human diseases, including neurological disorders and cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands (Csf1a, Csf1b, and Il34) in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors, Csf1ra and Csf1rb, however there is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Furthermore, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation, resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rbΔ4bp-deficient and il34Δ5bp-deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors, and the expression of csf1ra and csf1rb is nonoverlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis.

Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. Therapeutic targeting of the CSF1R pathway is a promising strategy in many human diseases, including neurological disorders or cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands - Csf1a, Csf1b and Il34, in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors as it is diminished in both csf1ra Δ5bp and csf1rb Δ 4bp mutants. There is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Further, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rb Δ4bp and il34 Δ5bp deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors and that the expression of csf1ra and csf1rb is non-overlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis. Key points: csf1ra and csf1rb are indispensable for macrophage differentiation and together with csf1a regulate embryonic macrophage fates in zebrafish csf1rb is important for granulocyte differentiation and migration and together with il34 it regulates embryonic granulocytic fates in zebrafish

Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kita(b5/b5) or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

PDMS microchambers and microchannels were evaluated in many studies of the specific in vitro cultivation. Modern microtopographical methods bring new possibilities how to prepare transparent planar blocks with isotropic lamellas. On the other hand, the planar nanofiber sheets are another type of modern culture substrate. Neonatal myocytes and the cardiac muscle cell lineage HL-1 were used for tests of the cell adherence, cell survival and cell orientation on pure or modified polydimethylsiloxane (PDMS) or polycaprolactone (PCL). PDMS with fibronectin coating was shown to be the material with the best cell adherence, viability and isotropic cell orientation from all tested variants.

Ischemic heart disease and resulting acute myocardial infarction (AMI) is one of the main causes of morbidity and mortality in industrial countries. The idea for the modern therapeutic strategy, which should activate the migration of stem/progenitor cells or reduce the migration of inflammatory cells in AMI regions, has emerged in the last 15 years, mainly as a result of physiological observation and post-mortem histology. Published data from direct measurements of cell migration are very limited. We prepared a universal set-up that can be used for the testing of cell migration in AMI micro-environment. Mesenchymal stromal cells (MSCs), the most commonly used stem/progenitor cells in experimental cellular therapy for AMI, were used in the recent set-up tests. The cells, which should be tested for their migration potential, were injected into the starting point in a special micro-chamber on the substrate, and optics of the microscope allowed a time-lapse recording of cells in micrometre resolution every 2 min. Our software tools provided precise 2D and 3D tracking of moving cells and data export for statistical analysis. Set-up should be upgraded to a fully-automatic preclinical screening tool in the future.

Hematopoiesis is a precisely orchestrated process regulated by the activity of hematopoietic cytokines and their respective receptors. Due to an extra round of whole genome duplication during vertebrate evolution in teleost fish, zebrafish have two paralogs of many important genes, including genes involved in hematopoiesis. Importantly, these duplication events brought increased level of complexity in such cases, where both ligands and receptors have been duplicated in parallel. Therefore, precise understanding of binding specificities between duplicated ligand-receptor signalosomes as well as understanding of their differential expression provide an important basis for future studies to better understand the role of duplication of these genes. However, although many recent studies in the field have partly addressed functional redundancy or sub-specialization of some of those duplicated paralogs, this information remains to be scattered over many publications and unpublished data. Therefore, the focus of this review is to provide an overview of recent findings in the zebrafish hematopoietic field regarding activity, role and specificity of some of the hematopoietic cytokines with emphasis on crucial regulators of the erythro-myeloid lineages.

Small fish species, such as zebrafish and medaka, are increasingly gaining popularity in basic research and disease modeling as a useful alternative to rodent model organisms. However, the tracking options for fish within a facility are rather limited. In this study, we present an aquatic species tracking database, Zebrabase, developed in our zebrafish research and breeding facility that represents a practical and scalable solution and an intuitive platform for scientists, fish managers, and caretakers, in both small and large facilities. Zebrabase is a scalable, cross-platform fish tracking database developed especially for fish research facilities. Nevertheless, this platform can be easily adapted for a wide variety of aquatic model organisms housed in tanks. It provides sophisticated tracking, reporting, and management functions that help keep animal-related records well organized, including a QR code functionality for tank labeling. The implementation of various user roles ensures a functional hierarchy and customized access to specific functions and data. In addition, Zebrabase makes it easy to personalize rooms and racks, and its advanced statistics and reporting options make it an excellent tool for creating periodic reports of animal usage and productivity. Communication between the facility and the researchers can be streamlined by the database functions. Finally, Zebrabase also features an interactive breeding history and a smart interface with advanced visualizations and intuitive color coding that accelerate the processes.

Optical noninvasive methods are modern tools in studying subcellular ion exchanges and corresponding membrane voltage alterations. In past decades, genetically encoded voltage indicators (GEVIs) received attention due to the low toxicity and enhanced detection limits. The very promising GEVI Accelerated Sensor of Action Potentials 1 (ASAP1) was expressed in HEK293 cells and extracellular stimulation was performed using a microelectrode-arrays integrated pulse generator. The results demonstrate high fluorescence response and changes, fast kinetics 1.5±0.3 ms, and enhanced amplitude of fluorescence response, even if the voltage sensor is not completely repolarized. The results bring verification of advantageous properties of ASAP1 and confirm a big potential of modern voltage sensitive dyes and their application in neural activity sensing where these properties play an important role. At the same time, our testing method showed as a very advantageous in voltage sensitivity dyes properties validation.

In current cell tissue engineering and cardiology, beating models with behavior and properties similar to humans are necessary. The cardiac muscle HL-1 cells, whose genes have a parent with adult atrial myocytes, serve as a suitable model. HL-1 cells were seeded on a 120-electrode microelectrode (MEA) chamber and transient transfection of Accelerated Sensor of Action Potentials 1 (ASAP1), a genetically encoded voltage indicator, was performed. Simultaneous electrical and optical recording of cardiac cell culture electrical activity was made when the cells started to produce spontaneous action potentials. Recording synchronization was made using TTL pulses. Results showed that HL-1 cells start to produce asynchronous spontaneous action potentials $(-375\pm 10\mu V)$ when reaching 90% MEA chamber confluency and which become periodical addition of extracellular calcium $(-501\pm 14\ \mu V,\ 2.1\pm 1.0\ Hz)$. The ASAP1's fluorescent response reached up to ${21}\pm{5}\%\ \Delta F/F$. The time constant between detection of electrical and fluorescent response $(\tau_{e/0})$ was determined as $12\pm 5$ ms.

A non-viral tool for the delivery of nucleic acids termed magnetofection was recently developed as a promising transgenic technique with high transfection efficiency for gene delivery into mammalian cells. Despite the fact that transfection efficiency was the objective in the past, the post-transfection cell morphology and the essential gigaseal formation between cells and patch clamp glass electrodes have not been studied in detail. The cell viability and fluorescent response of Accelerated Sensor of Action Potentials (ASAP1) were studied in somatic HEK293 cells with respect to preserving physiological cell behavior and morphology. The DNA vector (pcDNA3.1/Puro-CAG-ASAP1) was intracellularly delivered by DNA/polyethyleneimine/magnetic nanoparticles and the transfection protocols varied in complex formations were optimized with respect to transfection rate, cytotoxicity of modified nanoparticles and essential gigaseal formation needed for patch clamp technique. A patch clamp study of transfected cells was carried out 72 hours post-transfection. Our results showed the best complex formation in order DNA/magnetic nanoparticle/polyethyleneimine that provides 51.82% transfection efficiency, 83.45% of patch clamp applicable cells, and 90.15% of gigasealed patch clamp applicable cells. A significant difference in fluorescent response of transfected cells was not found compared to control. Thus, these observations suggested that a large amount of the cells were able to create a gigaseal with a glass electrode 72 hours from transfection despite the lower transfection efficiencies.

Gene delivery is a widespread strategy in current experimental medicine. In this work, we report a method for low-toxic intracellular DNA vector delivery and post transfection localisation of this vector in mouse embryonic fibroblast cell lines. The surface of modified ferumoxide nanoparticles conjugated with Rhoda-mine B isothiocyanate (FeNV-Rh) was modified with linear polyethyleneimine and medium molecular weight chitosan to increase Accelerated Sensor of Action Potentials DNA vector adhesion. The size of the FeNV-Rh/DNA transfection complex was studied using dynamic light scattering (DLS) and scanning electron microscopy (SEM) techniques. The transfection complex internalisation of plasmid expression and FeNV-Rh, and stability of rhodamine fluorescence in intracellular space were observed at time periods 6, 12, 24 and 48 h post transfection. Results showed high transfection complex intracellular biocompatibility—cell viability after Rh-MNP labelling was higher than 97% 24 h after transfection, and higher than 95% after the next 24 h. Selective FeNV-Rh localisation in the lysosomes was quantified. More than 82% of nanoparticles were localised in the lysosomes 12 h post transfection and 94% of lysosomes had a significant and long-term deposit of nanoparticles. DNA vector expression was visible in >65% of the cells and precise protein localisation on the cell membrane was confirmed using confocal microscopy.

Mutations in MECP2 cause Rett syndrome, a severe neurological disorder with autism-like features. Duplication of MECP2 also causes severe neuropathology. Both diseases display immunological abnormalities that suggest a role for MECP2 in controlling immune and inflammatory responses. Here, we used mecp2-null zebrafish to study the potential function of Mecp2 as an immunological regulator. Mecp2 deficiency resulted in an increase in neutrophil infiltration and upregulated expression of the pro- and anti-inflammatory cytokines Il1b and Il10 as a secondary response to disturbances in tissue homeostasis. By contrast, expression of the proinflammatory cytokine tumor necrosis factor alpha (Tnfa) was consistently downregulated in mecp2-null animals during development, representing the earliest developmental phenotype described for MECP2 deficiency to date. Expression of tnfa was unresponsive to inflammatory stimulation, and was partially restored by re-expression of functional mecp2 Thus, Mecp2 is required for tnfa expression during zebrafish development and inflammation. Finally, RNA sequencing of mecp2-null embryos revealed dysregulated processes predictive for Rett syndrome phenotypes.

The highly anisotropic interactions in organic semiconductors together with the soft character of organic materials lead to strong coupling between nuclear vibrations and exciton dynamics, which potentially results in anomalous electrical, optical and optoelectrical properties. Here, we report on the Raman antenna effect from organic semiconducting nanobelts 6,13-dichloropentacene (DCP), resulting from the coupling of molecular excitons and intramolecular phonons. The highly ordered crystalline structure in DCP nanobelts enables the precise polarization-resolved spectroscopic measurement. The angle-dependent Raman spectroscopy under resonant excitation shows that all Raman modes from the skeletal vibrations of DCP molecule act like a nearly perfect dipole antenna IRaman ∝ cos4(θ - 90), with almost zero (maximum) Raman scattering parallel (perpendicular) to the nanobelt's long-axis. The Raman antenna effect in DCP nanobelt is originated from the coupling between molecular skeletal vibrations and intramolecular exciton and the confinement of intermolecular excitons. It dramatically enhances the Raman polarization ratio (ρ = I‖/I⊥ > 25) and amplifies the anisotropy of the angle-dependent Raman scattering (κRaman = Imax/Imin > 12) of DCP nanobelts. These findings have crucial implications for fundamental understanding on the exciton-phonon coupling and its effects on the optical properties of organic semiconductors.

Fluorescence lifetime imaging (FLIM) is a modern optical method which increases the potential of standard microscopy. This paper shows the possibilities of extended fluorescence lifetime evaluation and imaging in studying three-dimensional structures such as compartments of living cells with different fluorescence lifetimes. The method for quasi-FLIM image calculation is presented and image processing steps useful for biological experiments are suggested. The method was tested on isolated cardiomyocyte cells (CMs) and rat bone marrow stromal cells (MSCs) labelled with SPIO-rhodamine nanoparticles and stained with standard fluorescent dyes. We proved it is possible to use an exponential decrease of fluorescence in time and lifetime parameters for pseudo-colour 3D image mapping of living cells and their compartments that is not a standard function of confocal microscopes.

Albumin plays an important role in human body. Its changed level in urine may indicate serious kidney disorders. We present a new point-of-care solution for sensitive detection of urine albumin - the miniature optical adapter for iPhone with in-built optical filters and a sample slot. The adapter exploits smart-phone flash to generate excitation light and camera to measure the level of emitted light. Albumin Blue 580 is used as albumin reagent. The proposed light-weight adapter can be produced at low cost using a 3D printer. Thus, the miniaturized detector is easy to use out of lab.

We employ ab initio calculations to investigate energetics of point defects in metastable rocksalt cubic Ta-N and Mo-N. Our results reveal a strong tendency to off-stoichiometry, i.e. defected structures are surprisingly predicted to be more stable than perfect ones with 1 : 1 metal-to-nitrogen stoichiometry. Despite the similarity of Ta-N and Mo-N systems in exhibiting this unusual behaviour, we also point out their crucial differences. While Ta-N significantly favours metal vacancies, Mo-N exhibits similar energies of formation regardless of the vacancy type (V-Mo, V-N) as long as their concentration is below approximate to 15 at.%. The overall lowest energies of formation were obtained for Ta0.78N and Mo0.91N, which are hence predicted to be the most stable compositions. To account for various experimental conditions during synthesis, we further evaluated the phase stability as a function of chemical potential of individual species. The proposed phase diagrams reveal four stable compositions, Mo0.84N, Mo0.91N, MoN0.69 and MoN0.44, in the case of Mo-N and nine stable compositions in the case of Ta-N indicating the important role of metal under-stoichiometry, since Ta0.75N and Ta0.78N significantly dominate the diagram. This is particularly important for understanding and designing experiments using non-equilibrium deposition techniques. Finally, we discuss the role of defect ordering and estimate a cubic lattice parameter as a function of defect contents and put them in the context of existing literature theoretical and experimental data.