Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy.

Fluorescence microscopy is limited by photoconversion due to continuous illumination, which results in not only photobleaching but also conversion of fluorescent molecules into species of different spectral properties through photoblueing. Here, we determined different fluorescence parameters of photoconverted products for various fluorophores under standard confocal and stimulated emission depletion (STED) microscopy conditions. We observed changes in both fluorescence spectra and lifetimes that can cause artifacts in quantitative measurements, which can be avoided by using exchangeable dyes.

Amphiphilic coumarin-based probes for live-cell STED nanoscopy of plasma membrane.

Plasma membranes are vital biological structures, serving as protective barriers and participating in various cellular processes. In the field of super-resolution optical microscopy, stimulated emission depletion (STED) nanoscopy has emerged as a powerful method for investigating plasma membrane-related phenomena. However, many applications of STED microscopy are critically restricted by the limited availability of suitable fluorescent probes. This paper reports on the development of two amphiphilic membrane probes, SHE-2H and SHE-2N, specially designed for STED nanoscopy. SHE-2N, in particular, demonstrates quick and stable plasma membrane labelling with negligible intracellular redistribution. Both probes exhibit outstanding photostability and resolution improvement in STED nanoscopy, and are also suited for two-photon excitation microscopy. Furthermore, microscopy experiments and cytotoxicity tests revealed no noticeable cytotoxicity of probe SHE-2N at concentration used for fluorescence imaging. Spectral analysis and fluorescence lifetime measurements conducted on probe SHE-2N using giant unilamellar vesicles, revealed that emission spectra and fluorescence lifetimes exhibited minimal sensitivity to lipid composition variations. These novel probes significantly augment the arsenal of tools available for high-resolution plasma membrane research, enabling a more profound exploration of cellular processes and dynamics.

New coumarin- and phenoxazine-based fluorescent probes for live-cell STED nanoscopy.

The potential of live-cell stimulated emission depletion (STED) nanoscopy has not yet been fully exploited. Currently, the main limitation is the small number of fluorophores and probes that can sustain high light intensity/high dose employed in STED. Namely, fluorophores suitable for STED nanoscopy must be bright and highly photostable and exhibit a large Stokes shift. To expand the list of available probes, we synthesized and evaluated several new membrane probes for live-cell STED nanoscopy. Of the tested probes, probes MePyr500, ThiaCN545 and NB640 not only allow high-resolution STED images, but also partition into the intracellular membranes relatively quickly, thus lacking the selectivity of labelling solely the plasma membrane. During experiments, cytotoxicity was observed merely with the probe ThiaCN545, which blebs the plasma membrane. In comparison with commercially available CellMask Orange and STAR RED (KK114) DPPE, all our tested probes exhibited better photostability with the exception of NB640, which had the fastest bleaching rate of all tested probes. The best overall results can be assigned to the probe MePyr500, providing high-resolution STED images as well as high photostability with no noticeable cytotoxicity, making it an excellent candidate for further development.

Nanoparticles Can Wrap Epithelial Cell Membranes and Relocate Them Across the Epithelial Cell Layer.

Although the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane's disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease.

Aerosol-Cell Exposure System Applied to Semi-Adherent Cells for Aerosolization of Lung Surfactant and Nanoparticles Followed by High Quality RNA Extraction.

Nanoparticle toxicity assessments have moved closer to physiological conditions while trying to avoid the use of animal models. An example of new in vitro exposure techniques developed is the exposure of cultured cells at the air-liquid interface (ALI), particularly in the case of respiratory airways. While the commercially available VITROCELL® Cloud System has been applied for the delivery of aerosolized substances to adherent cells under ALI conditions, it has not yet been tested on lung surfactant and semi-adherent cells such as alveolar macrophages, which are playing a pivotal role in the nanoparticle-induced immune response. OBJECTIVES: In this work, we developed a comprehensive methodology for coating semi-adherent lung cells cultured at the ALI with aerosolized surfactant and subsequent dose-controlled exposure to nanoparticles (NPs). This protocol is optimized for subsequent transcriptomic studies. METHODS: Semi-adherent rat alveolar macrophages NR8383 were grown at the ALI and coated with lung surfactant through nebulization using the VITROCELL® Cloud 6 System before being exposed to TiO2 NM105 NPs. After NP exposures, RNA was extracted and its quantity and quality were measured. RESULTS: The VITROCELL® Cloud system allowed for uniform and ultrathin coating of cells with aerosolized surfactant mimicking physiological conditions in the lung. While nebulization of 57 µL of 30 mg/mL TiO2 and 114 µL of 15 mg/mL TiO2 nanoparticles yielded identical cell delivered dose, the reproducibility of dose as well as the quality of RNA extracted were better for 114 µL.

How to control fluorescent labeling of metal oxide nanoparticles for artefact-free live cell microscopy.

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. This is best investigated using noninvasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labeling of nanoparticles with fluorescent dyes. However, as shown here, when the labeling is performed inadequately, unbound fluorescent dyes and inadvertently altered chemical and physical properties of the nanoparticles can result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we introduce a tested minimal combination of experimental methods to enable artefact-free fluorescent labeling of metal-oxide nanoparticles-the largest subpopulation of nanoparticles by industrial production and applications-and demonstrate its application in the case of TiO2 nanotubes. We (1) characterize potential changes of the nanoparticles' surface charge and morphology that might occur during labeling by using zeta potential measurements and transmission electron microscopy, respectively, and (2) assess stable binding of the fluorescent dye to the nanoparticles with either fluorescence intensity measurements or fluorescence correlation spectroscopy, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials' mechanism of action and will foster widespread and safe use of new nanomaterials.

Prediction of Chronic Inflammation for Inhaled Particles: the Impact of Material Cycling and Quarantining in the Lung Epithelium.

On a daily basis, people are exposed to a multitude of health-hazardous airborne particulate matter with notable deposition in the fragile alveolar region of the lungs. Hence, there is a great need for identification and prediction of material-associated diseases, currently hindered due to the lack of in-depth understanding of causal relationships, in particular between acute exposures and chronic symptoms. By applying advanced microscopies and omics to in vitro and in vivo systems, together with in silico molecular modeling, it is determined herein that the long-lasting response to a single exposure can originate from the interplay between the newly discovered nanomaterial quarantining and nanomaterial cycling between different lung cell types. This new insight finally allows prediction of the spectrum of lung inflammation associated with materials of interest using only in vitro measurements and in silico modeling, potentially relating outcomes to material properties for a large number of materials, and thus boosting safe-by-design-based material development. Because of its profound implications for animal-free predictive toxicology, this work paves the way to a more efficient and hazard-free introduction of numerous new advanced materials into our lives.