Super-resolution microscopy techniques enable optical imaging in live cells with unprecedented spatial resolution. They unfortunately lack the temporal resolution required to directly investigate ...cellular dynamics at scales sufficient to measure molecular diffusion. These fast time scales are, on the other hand, routinely accessible by spectroscopic techniques such as fluorescence correlation spectroscopy (FCS). To enable the direct investigation of fast dynamics at the relevant spatial scales, FCS has been combined with super-resolution stimulated emission depletion (STED) microscopy. STED-FCS has been applied in point or scanning mode to reveal nanoscale diffusion behavior of molecules in live cells. In this protocol, we describe the technical details of performing point STED-FCS (pSTED-FCS) and scanning STED-FCS (sSTED-FCS) measurements, from calibration and sample preparation to data acquisition and analysis. We give particular emphasis to 2D diffusion dynamics in cellular membranes, using molecules tagged with organic fluorophores. These measurements can be accomplished within 4-6 h by those proficient in fluorescence imaging.
Regulation of plasma membrane curvature and composition governs essential cellular processes. The material property of bending rigidity describes the energetic cost of membrane deformations and ...depends on the plasma membrane molecular composition. Because of compositional fluctuations and active processes, it is challenging to measure it in intact cells. Here, we study the plasma membrane using giant plasma membrane vesicles (GPMVs), which largely preserve the plasma membrane lipidome and proteome. We show that the bending rigidity of plasma membranes under varied conditions is correlated to readout from environment-sensitive dyes, which are indicative of membrane order and microviscosity. This correlation holds across different cell lines, upon cholesterol depletion or enrichment of the plasma membrane, and variations in cell density. Thus, polarity- and viscosity-sensitive probes represent a promising indicator of membrane mechanical properties. Additionally, our results allow for identifying synthetic membranes with a few well defined lipids as optimal plasma membrane mimetics.
Knowing the temperature distribution within the conducting walls of various multilayer-type materials is crucial for a better understanding of heat-transfer processes. This applies to many ...engineering fields, good examples being photovoltaics and microelectronics. In this work we present a novel fluorescence technique that makes possible the non-invasive imaging of local temperature distributions within a transparent, temperature-sensitive, co-doped Er:GPF1Yb0.5Er glass-ceramic with micrometer spatial resolution. The thermal imaging was performed with a high-resolution fluorescence microscopy system, measuring different focal planes along the
-axis. This ultimately enabled a precise axial reconstruction of the temperature distribution across a 500-µm-thick glass-ceramic sample. The experimental measurements showed good agreement with computer-modeled heat simulations and suggest that the technique could be adopted for the spatial analyses of local thermal processes within optically transparent materials. For instance, the technique could be used to measure the temperature distribution of intermediate, transparent layers of novel ultra-high-efficiency solar cells at the micron and sub-micron levels.
It is important to understand the nanomaterials intracellular trafficking and distribution and investigate their targeting into the nuclear area in the living cells. In our previous study, we firstly ...observed penetration of nonmodified positively charged carbon dots decorated with quaternary ammonium groups (QCDs) into the nucleus of mouse NIH/3T3 fibroblasts. Thus, in this work, we focused on deeper study of QCDs distribution inside two healthy mouse NIH/3T3 and L929 cell lines by fluorescence microspectroscopy and performed a comprehensive cytotoxic and DNA damage measurements. Real-time penetration of QCDs across the plasma cell membrane was recorded, concentration dependent uptake was determined and endocytic pathways were characterized. We found out that the QCDs concentration of 200 µg/mL is close to saturation and subsequently, NIH/3T3 had a different cell cycle profile, however, no significant changes in viability (not even in the case with QCDs in the nuclei) and DNA damage. In the case of L929, the presence of QCDs in the nucleus evoked a cellular death. Intranuclear environment of NIH/3T3 cells affected fluorescent properties of QCDs and evoked fluorescence blue shifts. Studying the intracellular interactions with CDs is essential for development of future applications such as DNA sensing, because CDs as DNA probes have not yet been developed.
Fluorescent carbon dots (CDs) are potential tools for the labeling of cells with many advantages such as photostability, multicolor emission, small size, rapid uptake, biocompatibility, and easy ...preparation. Affinity towards organelles can be influenced by the surface properties of CDs which affect the interaction with the cell and cytoplasmic distribution. Organelle targeting by carbon dots is promising for anticancer treatment; thus, intracellular trafficking and cytotoxicity of cationic CDs was investigated. Based on our previous study, we used quaternized carbon dots (QCDs) for treatment and monitoring the behavior of two human cancer cell MCF-7 and HeLa lines. We found similarities between human cancer cells and mouse fibroblasts in the case of QCDs uptake. Time lapse microscopy of QCDs-labeled MCF-7 cells showed that cells are dying during the first two hours, faster at lower doses than at higher ones. QCDs at a concentration of 100 µg/mL entered into the nucleus before cellular death; however, at a dose of 200 µg/mL, blebbing of the cellular membrane occurred, with a subsequent penetration of QCDs into the nuclear area. In the case of HeLa cells, the dose-depended effect did not happen; however, the labeled cells were also dying in mitosis and genotoxicity occurred nearly at all doses. Moreover, contrasted intracellular compartments, probably mitochondria, were obvious after 24 h incubation with 100 µg/mL of QCDs. The levels of reactive oxygen species (ROS) slightly increased after 24 h, depending on the concentration, thus the genotoxicity was likely evoked by the nanomaterial. A decrease in viability did not reach IC 50 as the DNA damage was probably partly repaired in the prolonged G0/G1 phase of the cell cycle. Thus, the defects in the G2/M phase may have allowed a damaged cell to enter mitosis and undergo apoptosis. The anticancer effect in both cell lines was manifested mainly through genotoxicity.
Bacterial infections acquired in healthcare facilities including hospitals, the so called healthcare acquired or nosocomial infections, are still of great concern worldwide and represent a ...significant economical burden. One of the major causes of morbidity is infection with Methicillin Resistant Staphylococcus aureus (MRSA), which has been reported to survive on surfaces for several months. Bactericidal activity of copper-TiO2 thin films, which release copper ions and are deposited on glass surfaces and heated to high temperatures, is well known even when illuminated with very weak UVA light of about 10 μW/cm2. Lately, there is an increased intrerest for one-dimensional TiO2 nanomaterials, due to their unique properties, low cost, and high thermal and photochemical stability. Here we show that copper doped TiO2 nanotubes produce about five times more ·OH radicals as compared to undoped TiO2 nanotubes and that effective surface disinfection, determined by a modified ISO 22196:2011 test, can be achieved even at low intensity UVA light of 30 μW/cm2. The nanotubes can be deposited on a preformed surface at room temperature, resulting in a stable deposition resistant to multiple washings. Up to 103 microorganisms per cm2 can be inactivated in 24 hours, including resistant strains such as Methicillin-resistant Staphylococcus aureus (MRSA) and Extended-spectrum beta-lactamase Escherichia coli (E. coli ESBL). This disinfection method could provide a valuable alternative to the current surface disinfection methods.
The submicron characterization of transient heat-transfer processes at solid–liquid interfaces is of great importance in many areas of science and engineering. This paper reports on a technique that ...allows for the transient thermal imaging of the temperature field underneath a growing bubble during nucleate boiling with submicron spatial resolution. The boiling experiments were performed on a temperature-sensitive, erbium-doped, heavy-metal glass, Er:ZBLALiP, used as a robust sensing material for the non-invasive, transient temperature measurements. These measurements were made by analyzing the intensity variations of the fluorescence emission. The thermal imaging of an active nucleation site was performed by utilizing high-resolution, fluorescence microscopy, which enabled a maximum spatial resolution of 370 nm/pixel. The high-speed acquisition above 400 fps ensured sampling of individual bubble-nucleation events. Our transient measurements clearly revealed temperature variations underneath the growing bubble, as well as a measurable bubble-departure frequency under saturated conditions. These encouraging results suggest the need for a systematic use of the corresponding fluorescence technique on enhanced boiling surfaces in order to define the local heat-transfer characteristics and to gain a better understanding of the underlying physical processes.
•Fluorescence thermal imaging of nucleate pool boiling process is presented.•Non-invasive imaging of transient-temperature fields at submicron spatial resolution.•Capturing of the individual microbubble-nucleation event is demonstrated.•The technique allows studies of various thermal processes at the submicron level.
Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from which it buds out. Previous studies have shown that the HIV-1 envelope is an ...environment of very low mobility, with the diffusion of incorporated proteins two orders of magnitude slower than in the plasma membrane. One of the reasons for this difference is thought to be the HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing, which has, until now, been difficult to assess experimentally. To further refine the model of the molecular mobility on the HIV-1 surface, we herein investigated the relative importance of membrane composition and curvature in simplified model membrane systems, large unilamellar vesicles (LUVs) of different lipid compositions and sizes (0.1⁻1 µm), using super-resolution stimulated emission depletion (STED) microscopy-based fluorescence correlation spectroscopy (STED-FCS). Establishing an approach that is also applicable to measurements of molecule dynamics in virus-sized particles, we found, at least for the 0.1⁻1 µm sized vesicles, that the lipid composition and thus membrane rigidity, but not the curvature, play an important role in the decreased molecular mobility on the vesicles' surface. This observation suggests that the composition of the envelope rather than the particle geometry contributes to the previously described low mobility of proteins on the HIV-1 surface. Our vesicle-based study thus provides further insight into the dynamic properties of the surface of individual HIV-1 particles, as well as paves the methodological way towards better characterisation of the properties and function of viral lipid envelopes in general.
Temperature is a critical parameter in biology, affecting the speed of reactions that occur in living systems. Nevertheless, measuring temperature with subcellular resolution (micrometric scale) and ...reliability remains a challenge to overcome. In this perspective, luminescence nanothermometry is a non‐contact technique which aims to measure temperature with a sub‐micrometric spatial resolution through the use of nanomaterials whose luminescence is affected solely by changes in temperature. Here, TiO2 nanoparticles doped with Eu+3 ions (Eu+3‐TiO2) are used for sensing temperature differences within single living cells. XRD, XPS, SEM, TEM and NEXAFS analysis allow the determination of the physicochemical characteristics of the Eu+3‐TiO2 nanoparticles and, the variation of the luminescence intensity of the Eu+3‐TiO2 nanoparticles with their temperature is investigated. The successful internalization of Eu+3‐TiO2 nanoparticles in different types of cells is observed. The luminescence of nanoparticles internalized in L929 fibroblast cells is measured when the system is heated in a biological relevant temperature range. Making use of an appropriate calibration curve the temperature variation inside the cells is determined with sensitivity of 0.5 K per 1% of luminosity change when heated.
Titanium oxide nanoparticles doped with Eu+3 ions are used to evaluate the temperature within single living cells. The luminescence of nanoparticles internalized in L929 fibroblast cells is measured when the system is heated in a biological relevant temperature range. Making use of a calibration curve the temperature variation inside cells is determined.
Do lipids tune B cell signaling? Urbančič, Iztok; Eggeling, Christian; Sezgin, Erdinc
Nature chemical biology,
06/2023, Letnik:
19, Številka:
6
Journal Article
Recenzirano
The role of lipid membrane domains in the activation of immune cells remains elusive. New microscopy data on B cell signaling support a mechanism in which lipid membrane domains consolidate upon B ...cell receptor clustering.
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