1,721,007 research outputs found
alpha-casein-membrane interactions: quantitative fluorescence analysis of the effects of protein aggregation state
No full textNile Red lifetime reveals microplastic identity
No full textMicroplastic pollution is recognized as a worldwide environmental problem. The increasing daily use and release of plastics into the environment have led to the accumulation of fragmented microplastics, with potentially awful consequences for the environment, and animal and human health. The detection and identification of microplastics are of utmost importance, but available methods are still limited. In this work, a new approach is presented for the analysis of microplastics based on hydrophobic fluorescence staining with Nile Red, using spectrally resolved confocal fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). Significant differences were observed in the emission spectra and fluorescence lifetimes of the analyzed microplastics. Nile Red fluorescence shows determinable behavior based on the polymer matrix and provides a fingerprint for the identification of fragments from different types of plastics. Lifetime imaging coupled with phasor analysis constitutes a fast, robust, and straightforward method for mapping and identifying different microplastics within the same sample in an aquatic environment
Microscopy method and apparatus for optical tracking of emitter objects
Full text linkMicroscopy method and apparatus for determining the posi tions of emitter objects in a three - dimensional space that comprises focusing scattered light or fluorescent light emitted by an emitter object, separating the focused beam in a first and a second optical beam, directing the first and the second optical beam through a varifocal lens having an optical axis such that the first optical beam impinges on the lens along the optical axis and the second beam impinges decentralized with respect to the optical axis of the varifocal lens , simultaneously capturing a first image created by the first optical beam and a second image created by the second optical beam , and determining the relative displacement of the position of the object in the first and in the second image , wherein the relative displacement contains the information of the axial position of the object along a perpendicular direction to the image plane
Two-Photon Imaging
No full textThis chapter will provide an overview of two-photon microscopy from elements of the theory underpinning fluorescence phenomena to functioning principles of a two-photon microscope including step-by-step practical advice on how to conduct an experiment using a two-photon microscope. In this context multi-photon excitation is also taken into consideration. By reading this chapter, you will have a synopsis of the basic principles of two-photon excitation, optical sectioning, and 3D microscopy. Furthermore, fundamentals of promising advanced methods for tissue imaging available for two-photon imaging as second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are briefly described together with classical applications on deep tissue imaging and functional brain imaging
Expansion and Light-Sheet Microscopy for Nanoscale 3D Imaging
No full textExpansion Microscopy (ExM) and Light-Sheet Fluorescence Microscopy (LSFM) are forefront imaging techniques that enable high-resolution visualization of biological specimens. ExM enhances nanoscale investigation using conventional fluorescence microscopes, while LSFM offers rapid, minimally invasive imaging over large volumes. This review explores the joint advancements of ExM and LSFM, focusing on the excellent performance of the integrated modality obtained from the combination of the two, which is refer to as ExLSFM. In doing so, the chemical processes required for ExM, the tailored optical setup of LSFM for examining expanded samples, and the adjustments in sample preparation for accurate data collection are emphasized. It is delve into various specimen types studied using this integrated method and assess its potential for future applications. The goal of this literature review is to enrich the comprehension of ExM and LSFM, encouraging their wider use and ongoing development, looking forward to the upcoming challenges, and anticipating innovations in these imaging techniques
Light Sheet Fluorescence Microscopy (LSFM) for Two-Photon Excitation Imaging of Thick Samples
Full text linkUnveiling water ordering in liquid–liquid phase separation using bovine serum albumin-polyethylene glycol systems
No full textLiquid–liquid phase separation (LLPS) is a fundamental physicochemical process where a homogeneous liquid solution spontaneously separates into two distinct liquid phases. Initially studied in polymer science, LLPS has emerged as a crucial mechanism in various biological processes, particularly in the formation of membrane-less organelles within cells. These organelles are biomolecular condensates that compartmentalise biochemical reactions without relying on traditional lipid membranes. LLPS is driven by a balance of enthalpic and entropic contributions, with protein–protein and protein-solvent interactions playing a pivotal role. Environmental factors, including temperature, pH, and solute concentrations, critically influence these interactions and thus the phase separation process. In this study, we investigate the role of water dynamics in the regulation of LLPS processes using a binary system of Bovine Serum Albumin (BSA) and Polyethylene Glycol (PEG). Classical spectroscopic methods and fluorescence lifetime imaging microscopy (FLIM), combined with phasor plot analysis, are employed to probe the local environment within protein condensates. Central to this approach is the use of 6-acetyl-2-dimethylaminonaphthalene (ACDAN), a fluorescent dye renowned for its sensitivity to water dipolar relaxation and changes in solvent polarity. This research aims to deepen our understanding of LLPS, particularly the role of water, offering insights into cellular processes and potential therapeutic strategies for LLPS-related diseases
α-casein micelles-membranes interaction: Flower-like lipid protein coaggregates formation
No full textBackground: Environmental conditions regulate the association/aggregation states of proteins and their action in cellular compartments. Analysing protein behaviour in presence of lipid membranes is fundamental for the comprehension of many functional and dysfunctional processes. Here, we present an experimental study on the interaction between model membranes and α-casein. α-casein is the major component of milk proteins and it is recognised to play a key role in performing biological functions. The conformational properties of this protein and its capability to form supramolecular structures, like micelles or irreversible aggregates, are key effectors in functional and pathological effects. Methods: By means of quantitative fluorescence imaging and complementary spectroscopic methods, we were able to characterise α-casein association state and the course of events induced by pH changes, which regulate the interaction of this molecule with membranes. Results: The study of these complex dynamic events revealed that the initial conformation of the protein critically regulates the fate of α-casein, size and structure of the newly formed aggregates and their effect on membrane structures. Disassembly of micelles due to modification in electrostatic interactions results in increased membrane structure rigidity which accompanies the formation of protein lipid flower-like co-aggregates with protein molecules localised in the external part. General significance: These results may contribute to the comprehension of how the initial state of a protein establishes the course of events that occur upon changes in the molecular environment. These events which may occur in cells may be essential to functional, pathological or therapeutical properties specifically associated to casein proteins
Laser-Fabricated Fluorescent, Ligand-Free Silicon Nanoparticles: Scale-up, Biosafety, and 3D Live Imaging of Zebrafish under Development
Full text linkThis work rationalizes the scalable synthesis of ultrasmall, ligand-free silicon nanomaterials via liquid-phase pulsed laser ablation process using picosecond pulses at ultraviolet wavelengths. Results showed that the irradiation time drives hydrodynamic NP size. Isolated, monodisperse Si-NPs are obtained at high yield (72%) using post-treatment process. The obtained Si-NPs have an average size of 10 nm (not aggregated) and display photoemission in the green spectral range. We directly characterized the ligand-free Si-NPs in a vertebrate animal (zebrafish) and assessed their toxicity during the development. In vivo assay revealed that Si-NPs are found inside in all the early life stages of embryos and larvae growth, showing that the biosafety of Si-NPs and malformation types are independent of the Si-NP dose. Si-NPs were directly imaged inside developing embryos by spinning disk-imaging technique with optical sectioning capability. We showed that Si-NPs can passively enter inside embryos by the pore canals of chorion, can diffuse in the circulatory system, i.e., blood vessel, and accumulate inside larvae midgut and yolk sac, and in the eye lens, indicating the crossing of the blood barrier
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