Setting up multicolour TIRF microscopy down to the single molecule level

Investigating biological mechanisms in ever greater detail requires continuous advances in microscopy techniques and setups. Total internal reflection fluorescence (TIRF) microscopy is a well-established technique for visualizing processes on the cell membrane. TIRF allows studies down to the single molecule level, mainly in single-colour applications. Instead, multicolour setups are still limited. Here, we describe our strategies for implementing a multi-channel TIRF microscopy system capable of simultaneous two-channel excitation and detection, starting from a single-colour commercial setup. First, we report some applications at high molecule density and then focus on the challenges we faced for achieving the single molecule level simultaneously in different channels, showing that rigorous optimizations on the setup are needed to increase its sensitivity up to this point, from camera setting to background minimization. We also discuss our strategies regarding crucial points of fluorescent labelling for this type of experiment: labelling strategy, kind of probe, efficiency, and orthogonality of the reaction, all of which are aspects that can influence the achievable results. This work may provide useful guidelines for setting up advanced single-molecule multi-channel TIRF experiments to obtain insights into interaction mechanisms on the cell membrane of living cells

Interplay between disorder and intersubband collective excitations in the two-dimensional electron gas

Intersubband absorption in modulation-doped quantum wells is usually appropriately described as a collective excitation of the confined two-dimensional electron gas. At sufficiently low electron density and low temperatures, however, the in-plane disorder potential is able to damp the collective modes by mixing the intersubband charge-density excitation with single-particle localized modes. Here we show experimental evidence of this transition. The results are analyzed within the framework of the density functional theory and highlight the impact of the interplay between disorder and the collective response of the two-dimensional electron gas in semiconductor heterostructures.Comment: 5 pages, 4 figures, RevTeX. Accepted for publication in Phys. Rev. B (Rapid. Comm.

Targeting Peptides: The New Generation of Targeted Drug Delivery Systems

Peptides can act as targeting molecules, analogously to oligonucleotide aptamers and antibodies. They are particularly efficient in terms of production and stability in physiological environments; in recent years, they have been increasingly studied as targeting agents for several diseases, from tumors to central nervous system disorders, also thanks to the ability of some of them to cross the blood–brain barrier. In this review, we will describe the techniques employed for their experimental and in silico design, as well as their possible applications. We will also discuss advancements in their formulation and chemical modifications that make them even more stable and effective. Finally, we will discuss how their use could effectively help to overcome various physiological problems and improve existing treatments

Observation of collapse of pseudospin order in bilayer quantum Hall ferromagnets

The Hartree-Fock paradigm of bilayer quantum Hall states with finite tunneling at filling factor $\nu$=1 has full pseudospin ferromagnetic order with all the electrons in the lowest symmetric Landau level. Inelastic light scattering measurements of low energy spin excitations reveal major departures from the paradigm at relatively large tunneling gaps. The results indicate the emergence of a novel correlated quantum Hall state at $\nu$=1 characterized by reduced pseudospin order. Marked anomalies occur in spin excitations when pseudospin polarization collapses by application of in-plane magnetic fields.Comment: ReVTeX4, 4 pages, 3 EPS figure

Metamorphosis of a Quantum Hall Bilayer State into a Composite Fermion Metal

Composite fermion metal states emerge in quantum Hall bilayers at total Landau level filling factor $\nu_T$=1 when the tunneling gap collapses by application of in-plane components of the external magnetic field. Evidence of this transformation is found in the continua of spin excitations observed by inelastic light scattering below the spin-wave mode at the Zeeman energy. The low-lying spin modes are interpreted as quasiparticle excitations with simultaneous changes in spin orientation and composite fermion Landau level index.Comment: 4 pages 4 figure

Soft Magnetorotons and Broken-Symmetry States in Bilayer Quantum Hall Ferromagnets

The recent report on the observation of soft magnetorotons in the dispersion of charge-density excitations across the tunneling gap in coupled bilayers at total Landau level filling factor $\nu_T=1$ is reviewed. The inelastic light scattering experiments take advantage of the breakdown of wave-vector conservation that occurs under resonant excitation. The results offer evidence that in the quantum Hall state there is a roton that softens and sharpens markedly when the phase boundary for transitions to highly-correlated compressible states is approached. These findings are interpreted with Hartree-Fock evaluations of the dynamic structure factor. The model includes the effect of disorder in the breakdown of wave-vector conservation and resonance enhancement profiles within a phenomenological approach. These results link the softening of magnetorotons to enhanced excitonic Coulomb interactions in the ferromagnetic bilayers.Comment: 6 pages, 5 figures; conference: EP2DS-1

An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes

Neurotrophins are growth factors of fundamental importance for the development, survival and maintenance of different neuronal and non-neuronal populations. Over the years, the use of labeled neurotrophins has helped in the study of their biological functions, leading to a better understanding of the processes that regulate their transport, traffic, and signaling. However, the diverse and heterogeneous neurotrophin labeling strategies adopted so far have often led to poorly reproducible protocols and sometimes conflicting conclusions. Here we present a robust, reliable, and fast method to obtain homogeneous preparations of fluorescent proNGF and NGF with 1:1 labeling stoichiometry. This strategy is well suited for several applications, ranging from advanced imaging techniques such as single particle tracking, to analyses that require large amounts of neurotrophins such as in vivo monitoring of protein biodistribution. As a proof of the quality of the labeled NGF and proNGF preparations, we provide a quantitative analysis of their colocalization with proteins involved in the signaling endosome function and sorting. This new analysis allowed demonstrating that proNGF localizes at a sub-population of endosomes not completely overlapped to the one hosting NGF

Observation of soft magnetorotons in bilayer quantum Hall ferromagnets

Inelastic light scattering measurements of low-lying collective excitations of electron double layers in the quantum Hall state at total filling nu_T=1 reveal a deep magnetoroton in the dispersion of charge-density excitations across the tunneling gap. The roton softens and sharpens markedly when the phase boundary for transitions to highly correlated compressible states is approached. The findings are interpreted with Hartree-Fock evaluations that link soft magnetorotons to enhanced excitonic Coulomb interactions and to quantum phase transitions in the ferromagnetic bilayers.Comment: ReVTeX4, 4 pages, 4 EPS figure

Mouse aortic muscle cells respond to oxygen following cytochrome P450 3A13 gene transfer

We have previously shown that a cytochrome P450 (CYP450) hemoprotein from the 3A subfamily CYP3A13 for the mouse, serves as the sensor in the contraction of the ductus arteriosus in response to increased oxygen tension. In addition, we have identified endothelin-1 (ET-1) as the effector for this response. Here, we examined whether Cyp3a13 gene transfer confers oxygen sensitivity to cultured muscle cells from mouse aorta. Coincidentally, we determined whether the same hemoprotein is normally present in the vessel. Cyp3a13-transfected aortic cells responded to oxygen, whereas no significant response was seen in native cells or in cells transfected with an empty vector. Furthermore, this oxygen effect was curtailed by the ET-1/ETA receptor antagonist BQ-123. We also found that CYP3A13 occurs naturally in aortic tissue and its isolated muscle cells in culture. We conclude that CYP3A13 is involved in oxygen sensing, and its action in the transfected muscle cells of the aorta, as in the native cells of the ductus, takes place through a linkage to ET-1. However, the response of aortic muscle to oxygen, conceivably entailing the presence of CYP3A13 at some special site, is not seen in the native situation, and may instead unfold upon transfection of the parent gene

Lysosome Dynamic Properties during Neuronal Stem Cell Differentiation Studied by Spatiotemporal Fluctuation Spectroscopy and Organelle Tracking

We investigated lysosome dynamics during neuronal stem cell (NSC) differentiation by two quantitative and complementary biophysical methods based on fluorescence: imaging-derived mean square displacement (iMSD) and single-particle tracking (SPT). The former extracts the average dynamics and size of the whole population of moving lysosomes directly from imaging, with no need to calculate single trajectories; the latter resolves the finest heterogeneities and dynamic features at the single-lysosome level, which are lost in the iMSD analysis. In brief, iMSD analysis reveals that, from a structural point of view, lysosomes decrement in size during NSC differentiation, from 1 \u3bcm average diameter in the embryonic cells to approximately 500 nm diameter in the fully differentiated cells. Concomitantly, iMSD analysis highlights modification of key dynamic parameters, such as the average local organelle diffusivity and anomalous coefficient, which may parallel cytoskeleton remodeling during the differentiation process. From average to local, SPT allows mapping heterogeneous dynamic responses of single lysosomes in different districts of the cells. For instance, a dramatic decrease of lysosomal transport in the soma is followed by a rapid increase of transport in the projections at specific time points during neuronal differentiation, an observation compatible with the hypothesis that lysosomal active mobilization shifts from the soma to the newborn projections. Our combined results provide new insight into the lysosome size and dynamics regulation throughout NSC differentiation, supporting new functions proposed for this organelle