Tick-borne encephalitis virus (TBEV) is a neurotropic orthoflavivirus that invades the central nervous system, leading to severe neurological manifestations. In this study, we developed a reporter virus comprising TurboGFP-expressing TBEV (tGFP-TBEV) as a versatile tool for advancing TBEV research. The tGFP-TBEV facilitates quantitative measurement of viral replication, enables precise tracking of individual infected cells, and supports high-throughput screening of potential antiviral compounds and virus-neutralization assays. Furthermore, tGFP-TBEV proved effective as a model for studying TBEV infection in rat organotypic cerebellar slices cultured ex vivo and for visualizing TBEV infection in the mouse brain. Using tissue-clearing protocols and light-sheet fluorescence microscopy, we achieved high-resolution, three-dimensional mapping of the TBEV distribution in the mouse brain. This analysis uncovered distinct patterns of TBEV tropism, with infections concentrated in regions associated with neurogenesis, olfactory processing, and specific neuroanatomical pathways. The ability to visualize infection at both the cellular and whole-organ level provides a new tool for detailed investigations into viral tropism, replication, and interactions with host tissues, paving the way for deeper insights into TBEV biology and the pathogenesis of tick-borne encephalitis.

• Klíčová slova
• TBEV, light-sheet microscopy, neurotropism, organotypic cerebellar slices, reporter viruses, tissue clearing,
• MeSH
• klíšťová encefalitida * virologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• mozek * virologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• replikace viru MeSH
• reportérové geny MeSH
• tropismus virů MeSH
• viry klíšťové encefalitidy * genetika   fyziologie   MeSH
• zobrazování trojrozměrné MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• luminescentní proteiny MeSH

The pH value is one of the most frequently measured chemical parameters, yet developing nanometric sensors capable of accurately mapping pH distribution and dynamics with high spatial and temporal resolution remains a significant challenge. Such sensors are vital for advancing our understanding of numerous physiological and pathological processes. Nanoparticle-based sensors, commonly referred to as nanosensors, represent a promising class of optical sensors, with fluorescence lifetime-based probes offering superior sensitivity and quantitative reliability. However, existing pH nanosensors relying on fluorescence lifetime are challenging to synthesize and often suffer from poor biocompatibility, narrow pH response ranges, low stability, and calibration-dependent performance. Here, we overcome these limitations by introducing a water-dispersible pH nanosensor based on fluorescence lifetime of colloidal carbon dots (CDs) derived via a one-step reaction from a single precursor Rhodamine B. These CDs are biocompatible, non-toxic, and stable in highly acidic/basic conditions, which makes them well-suited for intracellular applications. The intrinsic fluorescence lifetime of these CDs exhibits a pseudo-linear, self-referencing response across exceptionally broad pH range (1-11), driven by pH-induced transformations of their electronic structure occurring during protonation and deprotonation of CD surface. By applying micrometer-resolution, quantitative pH imaging via fluorescence lifetime imaging microscopy, we demonstrate how CDs are preferentially sequestered in lysosomes of human skin fibroblasts, enabling precise quantification of inhibitor-induced pH changes within these organelles. Our findings highlight a significant potential of the CD nanosensors for precise monitoring of lysosomal pH in living cells, offering broad utility in biomedical research and potential studies of pH-associated cellular dysfunction.

• Klíčová slova
• Carbon dots, Fluorescence, Fluorescence lifetime imaging microscopy, Intracellular sensing, pH nanosensor,
• Publikační typ
• časopisecké články MeSH

Marine ecosystems are in the spotlight, because environmental changes are threatening biodiversity and ecological functions. In this context, microalgae play key ecological roles both in planktonic and benthic ecosystems. Consequently, they are considered indispensable targets for global monitoring programs. However, due to their high spatial and temporal variability and to difficulties of species identification (still relying on microscopy observations), the assessment of roles played by these components of marine ecosystems is demanding. In addition, technologies for a 3D assessment of their complex morphology are scarcely available. Here, we present a comprehensive workflow for retrieving 3D information on microalgae with diverse geometries through holographic microscopy operating in flow-cytometry mode onboard a lab on a chip device. Depending on the rotation patterns of samples, a tailored approach is used to retrieve their rolling angles. We demonstrate the feasibility of measuring 3D data of various microalgae, contingent on the intrinsic optical properties of cells. Specifically, we show that for quasi-transparent and low-scattering microorganisms, the retrieved angles permit quantitative 3D tomographic refractive index (RI) mapping to be achieved, providing full characterization of the alga in terms of its inner structure and outer shape. Moreover, even in the most challenging scenarios, where microalgae exhibit high light absorption or strong scattering, quantitative 3D shape reconstructions of diatoms and dinoflagellates can be at least achieved. Finally, we compare our direct 3D measurements with 2D inferences of 3D properties, obtained using a commercially available microscopy system. The ability to non-invasively obtain 3D information on microalgae marks a fundamental advancement in the field, unlocking a wealth of novel biological insights for characterizing aquatic ecosystems.

• Publikační typ
• časopisecké články MeSH

BACKGROUND AND OBJECTIVE: Digital Holographic Microscopy provides a new kind of quantitative image data about live cells' in vitro activities. Apart from non-invasive and staining-free imaging, it offers topological weighting of cell mass. This led us to develop a particular tool for assessing cell mass dynamics. METHODS: Programming language Python and a training set of time-lapse images of adherent HT-1080 cells derived from human fibrosarcoma taken with dry objective 40x/0.95 at 30-second intervals were used to create the Analytical Image Differencing (AID) method. RESULTS: The AID makes the best of these new data by evaluating the difference between the chosen two quantitative phase images from the time-lapse series. The contribution of the method is demonstrated on hiQPI (Holographic Incoherent-light-source Quantitative Phase Imaging) image data taken with a Q-phase microscope. The analysis outputs are graphical and complemented with numerical data. To underscore the significance of the Analytical Image Differencing (AID) method, an initial pilot experiment was conducted to show the available analyses of sequential overlapping images capturing the movement of cancer cells. Notably, besides defining changes in areas used by the cell (newly or steadily occupied or better abandoned) it is an introduction to the zero-line concept, which denotes spots of tranquility among continuously moving surroundings. CONCLUSIONS: The measurement of zero-line length has emerged as a novel biomarker for characterizing cell mass transfer. The sensitivity of phase change measurements is demonstrated. The noise quality of input images obtained with incoherent (hiQPI) and coherent (QPI) methods is compared. The resulting effect on the AID method output is also shown. The findings of this study introduce a novel approach to evaluating cellular behavior in vitro. The concept emerged as a particularly noteworthy outcome. Collectively, these results highlight the substantial potential of AID in advancing the field of cancer cells biology, particularly.

• Klíčová slova
• Biophysics, Cancer cell migration, Digital holographic microscopy, Image processing, Live cell imaging, Non-invasive, Quantitative phase imaging, Staining-free imaging,
• MeSH
• algoritmy MeSH
• časosběrné zobrazování MeSH
• fibrosarkom patologie   MeSH
• holografie metody   MeSH
• lidé MeSH
• mikroskopie MeSH
• nádorové buněčné linie MeSH
• počítačové zpracování obrazu MeSH
• pohyb buněk * MeSH
• programovací jazyk MeSH
• software MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Monoclonal antibodies (mAb) are key therapeutic agents in cancer immunotherapy and exert their effects through Fc receptor-dependent and -independent mechanisms. However, the nanoscale receptor reorganization resulting from mAb binding and its implications for the therapeutic mode of action remain poorly understood. Here, we present a multi-target 3D RESI super-resolution microscopy technique that directly visualizes the structural organization of CD20 receptors and the Type I (e.g., Rituximab) and Type II (e.g., Obinutuzumab) anti-CD20 therapeutic antibodies and quantitatively analyze these interactions at single-protein resolution in situ. We discover that, while Type I mAbs promote higher-order CD20 oligomerization, Type II mAbs induce limited clustering, leading to differences in therapeutic function. Correlating RESI with functional studies for Type II antibodies with different hinge region flexibilities, we show that the oligomeric CD20 arrangement determines the Type I or Type II function. Thus, the nanoscale characterization of CD20-mAb complexes enhances our understanding of the structure-function relationships of therapeutic antibodies and offers insights into the design of next-generation mAb therapies.

• MeSH
• antigeny CD20 * imunologie   chemie   metabolismus   MeSH
• humanizované monoklonální protilátky * terapeutické užití   chemie   MeSH
• imunoterapie * metody   MeSH
• lidé MeSH
• monoklonální protilátky * chemie   terapeutické užití   MeSH
• multimerizace proteinu MeSH
• nádorové buněčné linie MeSH
• nádory * terapie   imunologie   MeSH
• rituximab * terapeutické užití   chemie   imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD20 * MeSH
• humanizované monoklonální protilátky * MeSH
• monoklonální protilátky * MeSH
• obinutuzumab MeSH Prohlížeč
• rituximab * MeSH

A voltammetric sandwich immunosensor based on gold nanoparticles (AuNPs) conjugates and specific immunoglobulins (IgG@AuNPs) has been developed for the quantitative determination of inactivated hepatitis A virus (HAV) in a vaccine. For the first time, copper nanoparticles (CuNPs) were catalytically reduced from copper sulfate on the surface of AuNPs conjugates by a chemical reducing agent, reduced nicotinamide adenine dinucleotide (NADH). Control experiments showed that copper was not formed in the absence of AuNPs on the electrode surface or in the absence of NADH. The AuNPs and IgG@AuNPs conjugates before and after the reduction of CuNPs were characterized by transmission electron microscopy (TEM). A carbon-based screen-printed electrode (SPE), functionalized with laser reduced graphene oxide (LRGO) to enhance the conductivity of its working surface, was employed as the solid substrate for immobilizing the receptor layer. This layer consisted of 'capture' antibodies derived from rabbits immunized with HAV. During the development of the electrochemical immunosensor, particular attention was paid to the optimization of the conditions for the deposition time of copper on the surface of the gold particles of the IgG@AuNPs conjugates and the concentration of the reducing agent NADH. The elemental composition and electronic state of the atoms on the surface of the electrodes modified with AuNPs before and after the reduction of CuNPs with NADH were investigated by X-ray photoelectron spectroscopy (XPS). This study demonstrates that the electrochemical immunosensor amplifies the analytical signal from copper electrooxidation in biosensor for detecting HAV in vaccines. Concurrently, it reduces immunoassay costs and viral antigen detection time relative to traditional enzyme-linked immunosorbent assay (ELISA) systems.

• Klíčová slova
• Copper nanoparticles, Electrochemical immunosensor, Enzyme immunoassay, Gold nanoparticles, Hepatitis a virus vaccine, Parallel line method,
• Publikační typ
• časopisecké články MeSH

Recent studies have demonstrated the applicability of the "single particle" mode in laser ablation-inductively coupled plasma-mass spectrometry to map and size particles simultaneously. The transport efficiency (TE) is an important parameter in this configuration and affects the detection of individual nanoparticles, reliability of nanoparticle characterization, and related applications. This study introduces a novel method for the precise determination of TE, based on counting upconversion nanoparticles from gels characterized by fluorescent microscopy. The method was found to be most suitable for the 2940-nm laser ablation system, achieving virtually quantitative nanoparticle desorption, with TE primarily governed by ablation cell design and aerosol transport efficiency. With the 213-nm laser, attention had to be paid to incomplete desorption and possible nanoparticle redeposition at low laser fluences to avoid variability in TE measurements. Finally, use of the 193-nm laser-induced nanoparticle disintegration, resulting in elevated baseline noise and lower sensitivity, which prevented the use of this approach for the determination of TE. This study highlights the versatility of the proposed method, while also identifying its limitations, in terms of wavelength and fluence.

• Publikační typ
• časopisecké články MeSH

This study investigates the impact of hydroxyapatite (HA) nanoparticles (NPs) on the cellular responses of poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds in bone tissue engineering applications. Three types of PLCL scaffolds were fabricated, varying in HANPs content. Saos-2 osteoblast-like cells (OBs) and THP-1-derived osteoclast-like cells (OCs) were co-cultured on the scaffolds, and cell proliferation was assessed using the MTS assay. The amount of double-stranded DNA (dsDNA) was quantified to evaluate cell proliferation. Expression levels of OBs and OCs markers were analyzed via quantitative polymerase chain reaction (qPCR) and the production of Collagen type I was visualized using confocal microscopy. Additionally, enzymatic activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP or ACP5) was measured to assess OB and OC function, respectively. Interestingly, despite the scaffold's structured character supporting the growth of the Saos-2 OBs and THP-1-derived OCs coculture, the incorporation of HANPs did not significantly enhance cellular responses compared to scaffolds without HANPs, except for collagen type I production. These findings suggest the need for further investigation into the potential benefits of HANPs in bone tissue engineering applications. Nevertheless, our study contributes valuable insights into optimizing biomaterial design for bone tissue regeneration, with implications for drug screening and material testing protocols.

• Klíčová slova
• PLCL, bone regeneration, hydroxyapatite, osteoblasts, osteoclasts, scaffold, tissue engineering,
• MeSH
• buněčné linie MeSH
• hydroxyapatit * chemie   MeSH
• kokultivační techniky MeSH
• lidé MeSH
• nanočástice * chemie   MeSH
• nanovlákna * chemie   MeSH
• osteoblasty cytologie   metabolismus   účinky léků   MeSH
• osteogeneze MeSH
• osteoklasty cytologie   metabolismus   účinky léků   MeSH
• polyestery * chemie   MeSH
• proliferace buněk účinky léků   MeSH
• regenerace kostí * účinky léků   MeSH
• THP-1 buňky MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hydroxyapatit * MeSH
• poly(lactic acid-co-epsilon-caprolactone) MeSH Prohlížeč
• polyestery * MeSH

In this study, we employed stimulated Raman scattering (SRS) microscopy, augmented with sum frequency generation, to characterize complex solid-state mixtures, containing many solid-state forms of the same compound, for the first time. Five crystalline forms and one amorphous form of lactose were characterized and resolved, including two more recently defined anhydrous solid-state forms. Additionally, the complex solid-state character of several commercially available pharmaceutical tableting and inhalation grades of lactose was profiled. The advanced multimodal label-free microscopy method enabled visualization of the distribution of the solid-state forms with submicron spatial resolution, including the detection of trace levels. In addition, quantitative solid-state compositions of the lactose products were estimated. Overall SRS microscopy allows sensitive and specific spatially resolved solid-state characterization of complex mixtures, beyond what is possible with established (nonspatially resolved) characterization methods.

• MeSH
• laktosa * chemie   analýza   MeSH
• léčivé přípravky chemie   MeSH
• mikroskopie MeSH
• nelineární optická mikroskopie * metody   MeSH
• Ramanova spektroskopie * metody   MeSH
• tablety chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• laktosa * MeSH
• léčivé přípravky MeSH
• tablety MeSH

Segmentation of macromolecular structures is the primary bottleneck for studying biomolecules and their organization with electron microscopy in 2D/3D - requiring months of manual effort. Transformer-based Rapid Dimensionless Instance Segmentation (TARDIS) is a deep learning framework that automatically and accurately annotates membranes and filaments. Pre-trained TARDIS models can segment electron tomography (ET) reconstructions from both 3D and 2D electron micrographs of cryo and plastic-embedded samples. Furthermore, by implementing a novel geometric transformer architecture, TARDIS is the only method to provide accurate instance segmentations of these structures. Reducing the annotation time for ET data from months to minutes, we demonstrate segmentation of membranes and filaments in over 13,000 tomograms in the CZII Data Portal. TARDIS thus enables quantitative biophysical analysis at scale for the first time. We show this in application to kinetochore-microtubule attachment and viral-membrane interactions. TARDIS can be extended to new biomolecules and applications and open-source at https://github.com/SMLC-NYSBC/TARDIS.

• Klíčová slova
• Actin, CNN, Cryo-EM, Cryo-ET, DIST, Filaments, Instance Segmentation, Membranes, Microtubules, Point Cloud, Segmentation, Semantic Segmentation, TARDIS, TEM EM/ET,
• Publikační typ
• časopisecké články MeSH
• preprinty MeSH