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Single particle tracking (SPT) is one of the most widely used tools in optical microscopy to evaluate particle mobility in a variety of situations, including cellular and model membrane dynamics. Recent technological developments, such as Interferometric Scattering microscopy, have allowed recording of long, uninterrupted single particle trajectories at kilohertz framerates. The resulting data, where particles are continuously detected and do not displace much between observations, thereby do not require complex linking algorithms. Moreover, while these measurements offer more details into the short-term diffusion behaviour of the tracked particles, they are also subject to the influence of localisation uncertainties, which are often underestimated by conventional analysis pipelines. we thus developed a Python library, under the name of TRAIT2D (Tracking Analysis Toolbox – 2D version), in order to track particle diffusion at high sampling rates, and analyse the resulting trajectories with an innovative approach. The data analysis pipeline introduced is more localisation-uncertainty aware, and also selects the most appropriate diffusion model for the data provided on a statistical basis. A trajectory simulation platform also allows the user to handily generate trajectories and even synthetic time-lapses to test alternative tracking algorithms and data analysis approaches. A high degree of customisation for the analysis pipeline, for example with the introduction of different diffusion modes, is possible from the source code. Finally, the presence of graphical user interfaces lowers the access barrier for users with little to no programming experience.

The high natural abundance of aluminium makes the respective fluorophores attractive for various optical applications, but photoluminescence quantum yields above 0.7 have yet not been reported for solutions of aluminium complexes. In this contribution, a dinuclear aluminium(III) complex featuring enhanced photoluminescence properties is described. Its facile one-pot synthesis originates from a readily available precursor and trimethyl aluminium. In solution, the complex exhibits an unprecedented photoluminescence quantum yield near unity (Φabsolute 1.0±0.1) and an excited-state lifetime of 2.3 ns. In the solid state, J-aggregation and aggregation-caused quenching are noted, but still quantum yields of 0.6 are observed. Embedding the complex in electrospun non-woven fabrics yields a highly fluorescent fleece possessing a quantum yield of 0.9±0.04.

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye., Single molecule tracking of fluorescent proteins in live cells is temporally limited by fluorophore photobleaching. Here the authors show using fluorophore pairs that FRET competes with photobleaching to improve photostability and allow longer-term tracking of both single proteins and complexes.

The process, how lipids are removed from the circulation and transferred from high density lipoprotein (HDL) – a main carrier of cholesterol in the blood stream – to cells, is highly complex. HDL particles are captured from the blood stream by the scavenger receptor, class B, type I (SR-BI), the so-called HDL receptor. The details in subsequent lipid-transfer process, however, have not yet been completely understood. The transfer has been proposed to occur directly at the cell surface across an unstirred water layer, via a hydrophobic channel in the receptor, or after HDL endocytosis. The role of the target lipid membrane for the transfer process, however, has largely been overlooked. Here, we studied at the single molecule level how HDL particles interact with synthetic lipid membranes. Using (high-speed) atomic force microscopy and fluorescence correlation spectroscopy (FCS) we found out that, upon contact with the membrane, HDL becomes integrated into the lipid bilayer. Combined force and single molecule fluorescence microscopy allowed us to directly monitor the transfer process of fluorescently labelled amphiphilic lipid probe from HDL particles to the lipid bilayer upon contact.

Indexación: Scopus. Gold nanorods are highly reactive, have a large surface-to-volume ratio, and can be functionalized with biomolecules. Gold nanorods can absorb infrared electromagnetic radiation, which is subsequently dispersed as local heat. Gold nanoparticles can be used as powerful tools for the diagnosis and therapy of different diseases. To improve the biological barrier permeation of nanoparticles with low cytotoxicity, in this study, we conjugated gold nanorods with cell-penetrating peptides (oligoarginines) and with the amphipathic peptide CLPFFD. Methods: We studied the interaction of the functionalized gold nanorods with biological membrane models (liposomes) by dynamic light scattering, transmission electron microscopy and the Langmuir balance. Furthermore, we evaluated the effects on cell viability and permeability with an MTS assay and TEM. Results and Discussion: The interaction study by DLS, the Langmuir balance and cryo-TEM support that GNR-Arg7 CLPFFD enhances the interactions between GNRs and biological membranes. In addition, cells treated with GNR-Arg7 CLPFFD internalized 80% more nanoparticles than cells treated with GNR alone and did not induce cell damage. Conclusion: Our results indicate that incorporation of an amphipathic sequence into oligoarginines for the functionalization of gold nanorods enhances biological membrane nanoparticle interactions and nanoparticle cell permeability with respect to nanorods functionalized with oligoarginine. Overall, functionalized gold nanorods with amphipathic arginine rich peptides might be candidates for improving drug delivery by facilitating biological barrier permeation. https://www.dovepress.com/improving-cell-penetration-of-gold-nanorods-by-using-an-amphipathic-ar-peer-reviewed-fulltext-article-IJN

Dipyrromethene difluoride-cholesterol (TopFluor-Cholesterol, TF-Chol) is a widely used cholesterol analogue due to its excellent fluorescence properties and considerable similarity with natural cholesterol in terms of membrane partitioning. However, the suitability of TF-Chol for detecting lysosomal cholesterol deposition has recently been questioned. Here, we highlight the fact that the method of lipid delivery and the analysis of time-point both affect the membrane distribution and labeling pattern of TF-Chol, similarly as with radiolabeled cholesterol. Lysosomal sterol accumulation characteristic to a lysosomal storage disease is most readily detected when the probe is introduced via the physiological route, i.e. as a sterol fatty acid ester in low-density lipoprotein particles. When administered to cells from solvent, lysosomal sterol sequestration becomes evident after an overnight equilibration between membranes. Non

Fluorescence correlation spectroscopy in combination with super-resolution stimulated emission depletion microscopy (STED-FCS) is a powerful tool to investigate molecular diffusion with sub-diffraction resolution. It has been of particular use for investigations of two dimensional systems like cell membranes, but has so far seen very limited applications to studies of three-dimensional diffusion. One reason for this is the extreme sensitivity of the axial (z) STED depletion pattern to optical aberrations. We present here an adaptive optics-based correction method that compensates for these aberrations and allows STED-FCS measurements in the cytoplasm of living cells.

Dendritic cells (DCs) are important antigen-presenting cells in the immune system. They are responsible for the recognition and internalization of pathogens, followed by the induction of antigen specific CD4+ and CD8+ T cell responses, but also for the maintenance of peripheral T cell tolerance.

The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. Therapeutic control of immune responses therefore relies on thorough elucidation of the molecular processes occurring at these interfaces. However, the detailed investigation of each component's contribution to the formation and regulation of the contact is hampered by the complexities of cell composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially in the use of advanced imaging technology. One approach that circumvents these problems is to establish in vitro systems that faithfully mimic immune cell interactions, but allow complexity to be ‘dialled-in’ as needed. Here, we present an in vitro system that makes use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system presented here is expected to be widely applicable., Summary: Immune cell–cell interactions are reconstituted in free-standing vesicles, allowing the spatiotemporal aspects of immune synapse formation to be investigated.

Cells rely on versatile diffusion dynamics in their plasma membrane. Quantification of this often heterogeneous diffusion is essential to the understanding of cell regulation and function. Yet such measurements remain a major challenge in cell biology, usually due to low sampling throughput, a necessity for dedicated equipment, sophisticated fluorescent label strategies, and limited sensitivity. Here, we introduce a robust, broadly-applicable statistical analysis pipeline for large scanning-fluorescence-correlation-spectroscopy data sets, which uncovers the nanoscale heterogeneity of the plasma membrane in living cells by differentiating free from hindered diffusion modes of fluorescent lipid and protein analogues.

There is now a substantial literature demonstrating the potential of surfactant stabilised microbubbles as drug delivery agents; particularly in combination with therapeutic ultrasound. Microbubbles provide a means of encapsulating drugs to avoid interaction with healthy tissue.

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

Giant unilamellar vesicles (GUVs) are well-established model systems for studying membrane structure and dynamics. Electroformation, also referred to as electro-swelling, is one of the most prevalent methods for producing GUVs, as it enables modulation of the lipid hydration process to form relatively mono-disperse, defect-free vesicles. Currently, however, it is relatively expensive and time-consuming compared to other methods. In this study, we demonstrate that 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) GUVs can be prepared readily at a fraction of the cost on stainless steel electrodes such as commercially available syringe needles, without any evidence of lipid oxidation or hydrolysis.

The antibody rituximab, which binds to the protein CD20 on the surface of B-cells, has been used to treat B-cell malignancies for several years. However, the molecular mechanisms underlying this treatment are not yet fully understood. One well-established rituximab-induced mechanism, natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), has recently been described to involve the polarisation of bound rituximab and CD20 to one side of the B-cell. B-cells polarised this way were cleared more efficiently by NK-cells, which led us to further investigate the cellular events involved in the polarisation process. Using optical microscopy on rituximab-treated cells, we have found that the rituximab/CD20-rich, polarised side accumulated mitochondria and actin, whereas the nucleus was reorganised to the opposite side of the cell. Depleting actin via different methods correlated with a decrease in rituximab, mitochondria, and nucleus polarisation, suggesting polarisation to be actin-dependent, active process that triggers intracellular rearrangement. The influence of these intracellular rearrangements on the efficiency of NK-cell-mediated clearance of B-cell malignancies remains open for future investigation.

Nitrogen-vacancy (NV) colour centres in diamond are attractive fluorescence emitters owing to their unprecedented photostability and superior applicability to spin manipulation and sub-diffraction far-field optical microscopy. However, some applications are limited by the co-occurrence of dark state population and optical excitation. In this paper, we use fluorescence microscopy and correlation spectroscopy on single negatively charged NV centres in type IIa bulk diamond to unravel the population kinetics of a >100 s long-lived dark state. The bright-dark state interconversion rates show a quadratic dependence on the applied laser intensity, which implies that higher excited states are involved. Depopulation of the dark state becomes less effective at wavelengths above 532 nm, resulting in a complete fluorescence switch-off at wavelengths >600 nm. This switch is reversible by the addition of shorter wavelengths. This behaviour can be explained by a model consisting of three dark and three bright states of different excitation levels, with the most efficient interconversion via the respective higher excited states. This model accounts for the nonlinear dark state and photoswitching kinetics, as well as for the decrease of the NV's fluorescence lifetime with excitation intensity and the strong dependence of fluorescence emission on excitation intensity. Unfortunately, our data do not give enough insight to allow us to assign the different states to specific electronic states known from the literature. Nevertheless, our observations allowed us to improve the recording of fluorescence images of single NV centres with sub-diffraction spatial resolution but they also have important implications for studying their spin states. © IOP Publishing Ltd and Deutsche Physikalische Gesellschaft.

We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 μm × 100 μm-sized fields of view in

Using a mode-locked titanium: sapphire laser at 700 nm for two-photon excitation we studied fluorescence bursts from individual coumarin 120 molecules in water and triacetin. Fluorescence lifetimes and multichannel scaler traces have been measured simultaneously. Due to the fact that scattered excitation light as well as Raman scattered photons can be suppressed by a short-pass filter a very low background level was achieved. To identify the fluorophore by its characteristic fluorescence lifetime the time-resolved fluorescence signals were analyzed by a maximum likelihood estimator. The obtained average fluorescence lifetimes (tau) av equals 4.8 +/- 1.2 ns for coumarin 120 in water and (tau) av equals 3.3 +/- 0.6 for coumarin 120 in triacetin are in good agreement with results obtained from separate measurements at higher concentrations. ©2005 Copyright SPIE - The International Society for Optical Engineering.