Laser-induced microinjury of the corneal basal epithelium and imaging of resident macrophage responses in a live, whole-eye preparation

The corneal epithelium is continuously subjected to external stimuli that results in varying degrees of cellular damage. The use of live-cell imaging approaches has facilitated understanding of the cellular and molecular mechanisms underlying the corneal epithelial wound healing process. Here, we describe a live, ex vivo, whole-eye approach using laser scanning confocal microscopy to simultaneously induce and visualize short-term cellular responses following microdamage to the corneal epithelium. Live-cell imaging of corneal cell layers was enabled using the lipophilic fluorescent dyes, SGC5 or FM4-64, which, when injected into the anterior chamber of enucleated eyes, readily penetrated and labelled cell membranes. Necrotic microdamage to a defined region (30 μm x 30 μm) through the central plane of the corneal basal epithelium was induced by continuously scanning for at least one minute using high laser power and was dependent on the presence of lipophilic fluorescent dye. This whole-mount live-cell imaging and microdamage approach was used to examine the behavior of Cx3cr1:GFP-expressing resident corneal stromal macrophages (RCSMs). In undamaged corneas, RCSMs remained stationary, but exhibited a constant extension and retraction of short (~5 μm) semicircular, pseudopodia-like processes reminiscent of what has previously been reported in corneal dendritic cells. Within minutes of microdamage, nearby anterior RCSMs became highly polarized and extended projections towards the damaged region. The extension of the processes plateaued after about 30 minutes and remained stable over the course of 2-3 hours of imaging. Retrospective immunolabeling showed that these responding RCSMs were MHC class II+. This study adds to existing knowledge of immune cell behavior in response to corneal damage and introduces a simple corneal epithelial microdamage and wound healing paradigm

Intra-arterial thrombolysis of occluded middle cerebral artery by use of collateral pathways in patients with tandem cervical carotid artery/middle cerebral artery occlusion

BACKGROUND AND PURPOSE: Cervical internal carotid artery (ICA) occlusion with middle cerebral artery (MCA) embolic occlusion is associated with a low rate of recanalization and poor outcome after intravenous thrombolysis. Prompt revascularization is required to prevent disabling stroke. We report our experience on acute ischemic stroke patients with tandem ICA or MCA occlusions treated with microcathether navigation and intra-arterial thrombolysis by use of collateral pathways including the posterior or anterior communicating arteries, or both pathways. MATERIALS AND METHODS: We retrospectively identified 8 patients with proximal ICA occlusion associated with MCA embolic occlusions treated with intra-arterial thrombolysis (IA rtPA). Access to the occluded MCA was obtained via catheter navigation through intact collateral pathways, including posterior communicating (PcomA) or anterior communicating (AcomA) arteries, without passing a microcathether through the acutely occluded ICA. We assessed clinical outcomes using modified Rankin scale (mRS) and National Institutes of Health Stroke Scale (NIHSS). RESULTS: Eight patients with a mean age of 57 ± 4 years and median NIHSS of 14 were identified. Mean time from stroke onset to intra-arterial thrombolysis was 292 ± 44 minutes. The MCA was revascularized completely in 5 of the 8 patients via collateral intra-arterial rtPA administration. All of the patients had a favorable outcome defined as a mRS of ≤2 or more at 1 and 3 months\u27 follow-up after thrombolytic therapy. One patient had an asymptomatic petechial hemorrhage. CONCLUSION: In this small number of patients with tandem occlusions of the ICA and MCA, intraarterial thrombolysis and recanalization of the MCA by use of collateral pathways to bypass the occluded ICA is a safe and efficacious therapeutic option

Distance Dependence of Electron Transfer Kinetics for Azurin Protein Adsorbed to Monolayer Protected Nanoparticle Film Assemblies

The distance dependence and kinetics of the heterogeneous electron transfer (ET) reaction for the redox protein azurin adsorbed to an electrode modified with a gold nanoparticle film are investigated using cyclic voltammetry. The nanoparticle films are comprised of nonaqueous nanoparticles, known as monolayer-protected clusters (MPCs), which are covalently networked with dithiol linkers. The MPC film assembly serves as an alternative adsorption platform to the traditional alkanethiolate self-assembled monolayer (SAM) modified electrodes that are commonly employed to study the ET kinetics of immobilized redox proteins, a strategy known as protein monolayer electrochemistry. Voltammetric analysis of the ET kinetics for azurin adsorbed to SAMs of increasing chain length results in quasi-reversible voltammetry with significant peak splitting. We observed rate constants (k°ET) of 12−20 s−1 for the protein at SAMs of shorter alkanethiolates that decays exponentially (β = 0.9/CH2 or 0.8/Å) at SAMs of longer alkanethiolates (9−11 methylene units) or an estimated distance of 1.23 nm and is representative of classical electronic tunneling behavior over increasing distance. Azurin adsorbed to the MPC film platforms of increasing thickness results in reversible voltammetry with very little voltammetric peaks splitting and nearly negligible decay of the ET rate over significant distances up to 20 nm. The apparent lack of distance dependence for heterogeneous ET reactions at MPC film assemblies is attributed to a two-step mechanism involving extremely fast electronic hopping through the MPC film architecture. These results suggest that MPC platforms may be used in protein monolayer electrochemistry to create adsorption platforms of higher architecture that can accommodate greater than monolayer protein coverage and increase the Faradaic signal, a finding with significant implications for amperometric biosensor design and development

On some related enumeration problems

The article is devoted to enumeration of partitioning of some classes of multisets, $n$-vertex regular graphs of the second degree and bitransversals of $n$-th order

AFM study of diamond particles internalization by monitoring MCF7 cell membrane stiffness changes

International audienceFluorescent nano-diamonds (fND) are attractive tools for nanoscale biological cellular imaging allowing both photoluminescence and magnetic resonance imaging [1]. Recent technological developments enable to fabricate bright fND particles of various sizes with high content of nitrogen-vacancy (NV) centres [2]. In this work we study internalization processes of NDs in the breast cancer MCF7 1DIV cell line using Atomic Force Microscopy imaging and force spectroscopy. fND particles of size range from 5 to 60 nm were used, prepared from Ib synthetic diamond, electron irradiated, annealed and plasma oxidized to create NV centers.Changes of cells stiffness were detected by AFM force measurements after introducing the fNDs into the cell medium. We observed an oscillating variation of cell membrane Young's modulus while the cells become stiffer. We believe that repetitive uptake/release processes of fNDs are responsible for these mechanical changes. Contrarywise, more confluent MCF7 cells (3DIV) did not show any significant change in Young’s Modulus, as compare with control ones. Indeed, it has been shown that differences in nanoparticle uptake can arise from how close the cells are to one another (confluence) [3] and how old they are.Moreover, our results suggest that studies on fNDs uptake should consider the cell cycle, as in a cell population, the dose of internalized nanoparticles in each cell varies as the expression of membrane proteins vary during the cell cycle [4]. The cell cycle is a series of events that lead to cell division and replication, consisting of four phases: G1 (when the cell increases its size), S (the cell synthesizes DNA), G2 (the cell synthesizes proteins for cell division) and M (the cell divides and the two daughter cells enter the G1 phase). During each event, cellular processes can vary; meaning that the rate at which a cell takes up foreign material, as for instance nanoparticles, may depend on the phase the cell is in [4-6], modifying accordingly their elasticity. When nanoparticle internalization is studied, it is therefore crucial to resolve how the state of the different cells affects the uptake. [1] L. Moore, M. Nesladek et al., Nanoscale (2014).[2] J. Havlik, M. Gulka, M. Nesladek et al., Nanoscale (2013).[3] B. Snijder, et al. Nature 461, 520–523 (2009). [4] E. Boucrot and T. Kirchhausen, Proc. Natl Acad. Sci. USA 104, 7939–7944 (2007). [5] D. Raucher and M. P. Sheetz, J. Cell Biol. 144, 497–506 (1999). 
[6] J. K. Schweitzer, E. E. Burke, H. V Goodson and C. D’Souza-Schorey. J. Biol. Chem. 280, 41628–41635 (2005)

Photoelectric detection of electron spin resonance of nitrogen-vacancy centres in diamond

The readout of negatively charged nitrogen-vacancy centre electron spins is essential for applications in quantum computation, metrology and sensing. Conventional readout protocols are based on the detection of photons emitted from nitrogen-vacancy centres, a process limited by the efficiency of photon collection. We report on an alternative principle for detecting the magnetic resonance of nitrogen-vacancy centres, allowing the direct photoelectric readout of nitrogen-vacancy centres spin state in an all-diamond device. The photocurrent detection of magnetic resonance scheme is based on the detection of charge carriers promoted to the conduction band of diamond by two-photon ionization of nitrogen-vacancy centres. The optical and photoelectric detection of magnetic resonance are compared, by performing both types of measurements simultaneously. The minima detected in the measured photocurrent at resonant microwave frequencies are attributed to the spin-dependent ionization dynamics of nitrogen-vacancy, originating from spin-selective non-radiative transitions to the metastable singlet state

Direct Structural Identification and Quantification of the Split-Vacancy Configuration for Implanted Sn in Diamond

We demonstrate formation of the ideal split-vacancy configuration of the Sn-vacancy center upon implantation into natural diamond. Using β^{-} emission channeling following low fluence ^{121}Sn implantation (2×10^{12}  atoms/cm^{2}, 60 keV) at the ISOLDE facility at CERN, we directly identified and quantified the atomic configurations of the Sn-related centers. Our data show that the split-vacancy configuration is formed immediately upon implantation with a surprisingly high efficiency of ≈40%. Upon thermal annealing at 920 °C ≈30% of Sn is found in the ideal bond-center position. Photoluminescence revealed the characteristic SnV^{-} line at 621 nm, with an extraordinarily narrow ensemble linewidth (2.3 nm) of near-perfect Lorentzian shape. These findings further establish the SnV^{-} center as a promising candidate for single photon emission applications, since, in addition to exceptional optical properties, it also shows a remarkably simple structural formation mechanism.status: publishe

Tailored internalization process of peptide coated luminescent nanodiamond particles in various cell lines.

International audienc

Exploiting ionization dynamics in the nitrogen vacancy center for rapid, high-contrast spin, and charge state initialization

We propose and experimentally demonstrate a method to strongly increase the sensitivity of spin measure-ments on nitrogen vacancy (NV) centers in diamond, which can be readily implemented in existing quantum sensing experiments. While charge state transitions of this defect are generally considered a parasitic effect to be avoided, we show here that these can be used to significantly increase the NV centers spin contrast, a key quantity for high-sensitivity magnetometry and high-fidelity state readout. The protocol consists of a two-step procedure, in which the charge state of the defect is first purified by a strong laser pulse, followed by weak illumination to obtain high spin polarization. We observe a relative improvement of the readout contrast by 17% and infer a reduction of the initialization error of more than 50%. The contrast enhancement is accompanied by a beneficial increase of the readout signal. For long sequence durations, typically encountered in high-resolution magnetometry, a measurement speedup by a factor of &amp;gt;1.5 is extracted, and we find that the technique is beneficial for sequences of any duration. Additionally, our findings give detailed insight into the charge and spin polarization dynamics of the NV center and provide actionable insights for direct optical, spin-to-charge, and electrical readout of solid-state spin centers.Funding Agencies|Austrian Science Fund (FWF) [G0A0520N, 101038045]; European Unions Horizon 2020 and Horizon Europe research and innovation programs; National Research, Development and Innovation Office in Hungary (NKFIH); EU QuantERA II MAESTRO project; Quantum Information National Laboratory via the Ministry of Culture and Innovation of Hungary; MTA Premium Postdoctoral Research Program; Knut and Alice Wallenberg Foundation through the WBSQD2 project; National Research, Development and Innovation Office in Hungary (NKFIH); FWO (Funds for Scientific Research) Flanders; European Unions Horizon 2020 research and innovation program; [I 3167-N27 SiC-EiC]; [864036]; [870002]; [877615]; [101046911]; [KKP129866]; [2018.0071]; [FK 137918]; [G0D1721N]</p