The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend ...on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes.
Spatial distribution and dynamics of plasma-membrane proteins are thought to be modulated by lipid composition and by the underlying cytoskeleton, which forms transient barriers to diffusion. So far ...this idea was probed by single-particle tracking of membrane components in which gold particles or antibodies were used to individually monitor the molecules of interest. Unfortunately, the relatively large particles needed for single-particle tracking can in principle alter the very dynamics under study. Here, we use a method that makes it possible to investigate plasma-membrane proteins by means of small molecular labels, specifically single GFP constructs. First, fast imaging of the region of interest on the membrane is performed. For each time delay in the resulting stack of images the average spatial correlation function is calculated. We show that by fitting the series of correlation functions, the actual protein “diffusion law” can be obtained directly from imaging, in the form of a mean-square displacement vs. time-delay plot, with no need for interpretative models. This approach is tested with several simulated 2D diffusion conditions and in live Chinese hamster ovary cells with a GFP-tagged transmembrane transferrin receptor, a well-known benchmark of membrane-skeleton–dependent transiently confined diffusion. This approach does not require extraction of the individual trajectories and can be used also with dim and dense molecules. We argue that it represents a powerful tool for the determination of kinetic and thermodynamic parameters over very wide spatial and temporal scales.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The relevant length scales in sub‐nanometer amplitude surface acoustic wave‐driven acoustic streaming are demonstrated. We demonstrate the absence of any physical limitations preventing the ...downscaling of SAW‐driven internal streaming to nanoliter microreactors and beyond by extending SAW microfluidics up to operating frequencies in the GHz range. This method is applied to nanoliter scale fluid mixing.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
We demonstrate high-temperature thermoelectric conversion in InAs/InP nanowire quantum dots by taking advantage of their strong electronic confinement. The electrical conductance G and the ...thermopower S are obtained from charge transport measurements and accurately reproduced with a theoretical model accounting for the multilevel structure of the quantum dot. Notably, our analysis does not rely on the estimate of cotunnelling contributions, since electronic thermal transport is dominated by multilevel heat transport. By taking into account two spin-degenerate energy levels we are able to evaluate the electronic thermal conductance K and investigate the evolution of the electronic figure of merit ZT as a function of the quantum dot configuration and demonstrate ZT ≈ 35 at 30 K, corresponding to an electronic efficiency at maximum power close to the Curzon–Ahlborn limit.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Synthesis and targeted delivery of dendrimer-based fluorescent biosensors in living HeLa cells are reported. Following electroporation dendrimers are shown to display specific subcellular ...localization depending on their size and surface charge and this property is preserved when they are functionalized with sensing moieties. We analyze the case of double dendrimer conjugation with pH-sensitive and pH-insensitive molecules leading to the realization of ratiometric pH sensors that are calibrated in vitro and in living cells. By tuning the physicochemical properties of the dendrimer scaffold sensors can be targeted to specific cellular compartments allowing selective pH measurements in different organelles in living cells. In order to demonstrate the modularity of this approach we present three different pH sensors with tuned H+ affinity by appropriately choosing the pH-sensitive dye. We argue that the present methodology represents a general approach toward the realization of targetable ratiometric sensors suitable to monitor biologically relevant ions or molecules in living cells.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
The ability of dendrimers to cross cell membranes is of much interest for their application in drug and gene delivery. Recent studies demonstrate that dendrimers are capable to enter cells by ...endocytosis, but the intracellular pathway following their internalization remains controversial. In this study we use confocal fluorescence microscopy to elucidate the intracellular trafficking properties of PAMAM dendrimers with high spatial and temporal resolution in living HeLa cells. Macromolecules of different chemical functionality (neutral, cationic and lipidated), size (from G2 up to G6) and surface charge are investigated and their internalization properties correlated with the molecular structure. Toxicity and internalization data are discussed that allow the identification of dendrimers maximizing intracellular uptake with the minimum effect on cell viability. Time-lapse imaging and colocalization assays with fluorescent biomarkers for endocytic vesicles demonstrate that dendrimers are internalized by both clathrin-dependent endocytosis and macropinocytosis and are eventually delivered to the lysosomal compartment. Moreover we analyzed the uptake of dendrimers in additional cell lines of practical interest for therapeutic purposes. These measurements together with a direct comparison with TAT peptides demonstrate that PAMAM dendrimers possess similar properties to these widely used cell-penetrating peptides and thanks to their chemical tunability may represent a valid alternative for drug and gene delivery.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Intracellular chloride (Cl⁻i) and pH(pHi) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of ...the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of Cl⁻i and pHi, but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl⁻ and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of Cl⁻i and pHi at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in Cl⁻i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and Cl⁻i in response to hypercapnia and seizure activity.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Fast and controllable surface acoustic wave (SAW) driven digital microfluidic temperature changes are demonstrated. Within typical operating conditions, the direct acoustic heating effect is shown to ...lead to a maximum temperature increase of about 10 °C in microliter water droplets. The importance of decoupling droplets from other on‐chip heating sources is demonstrated. Acoustic‐heating‐driven temperature changes reach a highly stable steady‐state value in ≈3 s, which is an order of magnitude faster than previously published. This rise time can even be reduced to ≈150 ms by suitably tailoring the applied SAW‐power excitation profile. Moreover, this fast heating mechanism can lead to significantly higher temperature changes (over 40 °C) with higher viscosity fluids and can be of much interest for on‐chip control of biological and/or chemical reactions.
Fast and controllable surface acoustic wave (SAW) driven digital microfluidic temperature changes are demonstrated. Small temperature changes in typical SAW microfluidic conditions and the possibility for rapid and controllable high temperature changes, for use with lab‐on‐a‐chip devices, are shown.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Abstract Human islets of Langerhans are composed mostly of glucagon-secreting α cells and insulin-secreting β cells closely intermingled one another. Current methods for identifying α and β cells ...involve either fixing islets and using immunostaining or disaggregating islets and employing flow cytometry for classifying α and β cells based on their size and autofluorescence. Neither approach, however, allows investigating the dynamic behavior of α and β cells in a living and intact islet. To tackle this issue, we present a machine-learning-based strategy for identification α and β cells in label-free infrared micrographs of living human islets without immunostaining. Intrinsic autofluorescence is stimulated by infrared light and collected both in intensity and lifetime in the visible range, dominated by NAD(P)H and lipofuscin signals. Descriptive parameters are derived from micrographs for ~ 10 3 cells. These parameters are used as input for a boosted decision-tree model (XGBoost) pre-trained with immunofluorescence-derived cell-type information. The model displays an optimized-metrics performance of 0.86 (i.e. area under a ROC curve), with an associated precision of 0.94 for the recognition of β cells and 0.75 for α cells. This tool promises to enable longitudinal studies on the dynamic behavior of individual cell types at single-cell resolution within the intact tissue.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Early diagnosis is one of the most important factors in determining the prognosis in cancer. Sensitive detection and quantification of tumour-specific biomarkers have the potential to improve ...significantly our diagnostic capability. Here, we introduce a triggerable aptamer-based nanostructure based on an oligonucleotide/gold nanoparticle architecture that selectively disassembles in the presence of the biomarker of interest; its optimization is based also on in-silico determination of the aptamer nucleotides interactions with the protein of interest. We demonstrate this scheme for the case of Prostate Specific Membrane Antigen (PSMA) and PSMA derived from PSMA-positive exosomes. We tested the disassembly of the system by diameter and count rate measurements in dynamic light scattering, and by inspection of its plasmon resonance shift, upon addition of PSMA, finding appreciable differences down to the sub-picomolar range; this points towards the possibility that this approach may lead to sensors competitive with diagnostic biochemical assays that require enzymatic amplification. More generally, this scheme has the potential to be applied to a broad range of pathologies with specific identified biomarkers.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
PDF
