1,721,124 research outputs found
Spatiotemporal fluctuation analysis of molecular diffusion laws in live-cell membranes
No full textA present challenge of membrane biophysics is deciphering the dynamic behavior of molecules, such as lipids and proteins, within the natural environment of a living-cell membrane. Here, a fluorescence fluctuation-based approach will be described, which makes it possible to probe the âdiffusion lawâ of molecules directly from imaging, in the form of a mean square displacement vs time-delay plot (iMSD), with no need for interpretative models. Of note, the presented approach does not require extraction of the molecular trajectories nor the use of bright fluorophores. Conversely, it can be used at high fluorophore density and with relatively dim fluorophores, such as GFP-tagged molecules transiently expressed within cells. The ability of this approach to resolve average molecular dynamic properties well below the diffraction limit will be discussed. Overall, this novel approach is proposed as a powerful tool for the determination of kinetic and thermodynamic parameters over wide spatial and temporal scales
Time-resolved biophysical approaches to nucleocytoplasmic transport
Full text linkMolecules are continuously shuttling across the nuclear envelope barrier that separates the nucleus from the cytoplasm. Instead of being just a barrier to diffusion, the nuclear envelope is rather a complex filter that provides eukaryotes with an elaborate spatiotemporal regulation of fundamental molecular processes, such as gene expression and protein translation. Given the highly dynamic nature of nucleocytoplasmic transport, during the past few decades large efforts were devoted to the development and application of time resolved, fluorescence-based, biophysical methods to capture the details of molecular motion across the nuclear envelope. These methods are here divided into three major classes, according to the differences in the way they report on the molecular process of nucleocytoplasmic transport. In detail, the first class encompasses those methods based on the perturbation of the fluorescence signal, also known as ensemble-averaging methods, which average the behavior of many molecules (across many pores). The second class comprises those methods based on the localization of single fluorescently-labelled molecules and tracking of their position in space and time, potentially across single pores. Finally, the third class encompasses methods based on the statistical analysis of spontaneous fluorescence fluctuations out of the equilibrium or stationary state of the system. In this case, the behavior of single molecules is probed in presence of many similarly-labelled molecules, without dwelling on any of them. Here these three classes, with their respective pros and cons as well as their main applications to nucleocytoplasmic shuttling will be briefly reviewed and discussed. Keywords: Fluorescence recovery after photobleaching, Single particle tracking, Fluorescence correlation spectroscopy, Diffusion, Transport, GFP, Nuclear pore complex, Live cell, Confocal microscop
Back to the Future: Genetically Encoded Fluorescent Proteins as Inert Tracers of the Intracellular Environment
Full text linkOver the past decades, the discovery and development of genetically encoded fluorescent proteins (FPs) has brought a revolution into our ability to study biologic phenomena directly within living matter. First, FPs enabled fluorescence-labeling of a variety of molecules of interest to study their localization, interactions and dynamic behavior at various scales—from cells to whole organisms/animals. Then, rationally engineered FP-based sensors facilitated the measurement of physicochemical parameters of living matter—especially at the intracellular level, such as ion concentration, temperature, viscosity, pressure, etc. In addition, FPs were exploited as inert tracers of the intracellular environment in which they are expressed. This oft-neglected role is made possible by two distinctive features of FPs: (i) the quite null, unspecific interactions of their characteristic β-barrel structure with the molecular components of the cellular environment; and (ii) their compatibility with the use of time-resolved fluorescence-based optical microscopy techniques. This review seeks to highlight the potential of such unique combinations of properties and report on the most significative and original applications (and related advancements of knowledge) produced to date. It is envisioned that the use of FPs as inert tracers of living matter structural organization holds a potential for several lines of further development in the next future, discussed in the last section of the review, which in turn can lead to new breakthroughs in bioimaging
Phasor identifier : a cloud-based analysis of Phasor-FLIM data on Python notebooks
Full text linkThis paper introduces an innovative approach utilizing Google Colaboratory (Colab) for the versatile analysis of phasor Fluorescence Lifetime Imaging Microscopy (FLIM) data collected from various samples (e.g., cuvette, cells, tissues) and in various input file formats. In fact, phasor-FLIM widespread adoption has been hampered by complex instrumentation and data analysis requirements. We mean to make advanced FLIM analysis more accessible to researchers through a cloud-based solution that i) harnesses robust computational resources, ii) eliminates hardware limitations, iii) supports both CPU and GPU processing, We envision a paradigm shift in FLIM data accessibility and potential, aligning with the evolving field of AI-driven FLIM analysis. This approach simplifies FLIM data handling and opens doors for diverse applications, from studying cellular metabolism to investigating drug encapsulation, benefiting researchers across multiple domains. The comparative analysis of freely distributed FLIM tools highlights the unique advantages of this approach in terms of adaptability, scalability, and open-source nature.This paper introduces an innovative approach utilizing Google Colaboratory for the versatile analysis of phasor fluorescence lifetime imaging microscopy (FLIM) data collected from various samples (e.g., cuvette, cells, tissues) and in various input file formats. In fact, phasor-FLIM widespread adoption has been hampered by complex instrumentation and data analysis requirements. We mean to make advanced FLIM analysis more accessible to researchers through a cloud-based solution that 1) harnesses robust computational resources, 2) eliminates hardware limitations, and 3) supports both CPU and GPU processing. We envision a paradigm shift in FLIM data accessibility and potential, aligning with the evolving field of artificial intelligence-driven FLIM analysis. This approach simplifies FLIM data handling and opens doors for diverse applications, from studying cellular metabolism to investigating drug encapsulation, benefiting researchers across multiple domains. The comparative analysis of f..
Nanoscopy on drug-encapsulating nanosystems by phasor-based fluorescence lifetime analysis
Full text linkNanoparticelle lipidiche multicomponenti ad alta fusogenicità cellulare per la vaccinazione a DNA e relativo processo di preparazione
No full textLa presente invenzione riguarda il processo di preparazione di nanoparticelle lipidiche multicomponenti (LNP) con elevata fusogenicità cellulare per la veicolazione di acidi nucleici e in particolare per la vaccinazione a DNA. L'invenzione riguarda anche le nanoparticelle così ottenute. In particolare, le nanoparticelle lipidiche dell'invenzione sono ottenute combinando lipidi cationici e lipidi ionizzabili in un rapporto specifico tra loro per essere somministrate sia per via sottocutanea (intradermica o intramuscolare) sia per via sistemica con minori effetti collaterali rispetto alle nanoparticelle lipidiche note nell'arte.The present invention concerns the process of preparing multicomponent lipid nanoparticles (LNPs) with high cellular futurogenicity for the delivery of nucleic acids and in particular for DNA vaccination. The invention also concerns the nanoparticles thus obtained. In particular, the lipid nanoparticles of the invention are obtained by combining cationic lipids and ionizable lipids in a specific ratio between them to be administered both subcutaneously (intradermal or intramuscular) and systemically with fewer side effects compared to the lipid nanoparticles known in the art
Spatiotemporal fluorescence correlation spectroscopy of inert tracers: a journey within cells, one molecule at a time
No full textIn Vivo Imaging of Single-Molecule Translocation through Nuclear Pore Complexes by Pair Correlation Functions
Full text linkNuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. Although there are many studies that look at the traffic in the nucleus and through the nuclear envelope we propose a method to detect the nucleocytoplasmic transport kinetics in an unperturbed cell, with no requirement for specific labeling of isolated molecules and, most important, in the presence of the cell milieu.The pair correlation function method (pCF) measures the time a molecule takes to migrate from one location to another within the cell in the presence of many molecules of the same kind. The spatial and temporal correlation among two arbitrary points in the cell provides a local map of molecular transport, and also highlights the presence of barriers to diffusion with millisecond time resolution and spatial resolution limited by diffraction. We use the pair correlation method to monitor a model protein substrate undergoing transport through NPCs in living cells, a biological problem in which single particle tracking (SPT) has given results that cannot be confirmed by traditional single-point FCS measurements because of the lack of spatial resolution.We show that obstacles to molecular flow can be detected and that the pCF algorithm can recognize the heterogeneity of protein intra-compartment diffusion as well as the presence of barriers to transport across NE
Capturing directed molecular motion in the nuclear pore complex of live cells
Full text linkNuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleo-cytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to tackle this issue in intact cells by an established combination of fluorescence correlation spectroscopy and real-time tracking of the center of mass of single NPCs. We find the dynamics of nucleoporin Nup153 to be regulated so as to produce rapid, discrete exchange between two separate positions within the NPC. A similar behavior is also observed for both karyopherinÎ21 transport-receptor and cargoes destined to nuclear import. Thus, we argue that directed Nup-mediated molecular motion may represent an intrinsic feature of the overall selective gating through intact NPCs
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