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Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin β1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs.

Hyperpolarizability is a measure of the nonlinear optical characteristics of natural or meta-atoms describing how the atoms become nonlinearly polarized by the induced local-field. However, determining hyperpolarizability in the case of structured plasmonic meta-atoms is not straightforward due to their relatively larger sizes, unique shapes, and the index of refraction of the surrounding dielectric medium. Also, the order-of-magnitude of hyperpolarizability may vary with the frequency of light especially when inter-band transitions in metals become dominant. Here, we experimentally and theoretically estimated the order-of-magnitude of the 1st-order hyperpolarizability of gold meta-atoms that can be used in designing nonlinear metasurfaces.

Plasmonic metasurfaces are promising as enablers of nanoscale nonlinear optics and flat nonlinear optical components. Nonlinear optical responses of such metasurfaces are determined by the nonlinear optical properties of individual nanostructured plasmonic meta-atoms, which are the building blocks of the metasurfaces. Unfortunately, no simple methods exist to determine the nonlinear coefficients (hyperpolarizabilities) of the meta-atoms hindering designing of nonlinear metasurfaces. Here, we develop the equivalent RLC circuit model of such meta-atoms to estimate their second-order nonlinear optical parameter i.e. the first-order hyperpolarizability in the optical spectral range. In parallel, we extract from second-harmonic generation experiments the spectrum of the 1st-order hyperpolarizabilities of individual meta-atoms consisting of asymmetrically shaped (elongated) plasmonic nanoprisms. Moreover, we verify our results using nonlinear hydrodynamic-FDTD and with calculations based on nonlinear scattering theory. All three approaches: analytical, experimental, and computational, yield results that agree very well. Our empirical RLC model can thus be used as a simple tool to enable efficient design of nonlinear plasmonic metasurfaces.

We experimentally measure the microscopic 2nd-order nonlinear optical response of plasmonic meta-atoms known as the 1st-order hyperpolarizability. This result is crucial to design nonlinear metasurfaces consisting of plasmonic meta-atoms.

Here, we experimentally determine the order-of-magnitude value of the 1st-order hyperpolarizability of plasmonic meta-atoms in metasurfaces. This result will prove useful in designing nonlinear metasurfaces for a variety of applications.

Optical microscopy is a standard tool to study biological objects. Diffraction limits the achievable lateral resolution of an optical microscope to around 200 nm restricting its applicability. However, recent advances in optical super-resolution techniques have shown that the diffraction does not impose a fundamental limit to resolution and can be circumvented. These super-resolution techniques can provide resolution approaching the nanometer scale and are enabling studies of biological objects with unprecedented capabilities. However, most of the super-resolution techniques are based on using fluorescent dyes, complicating their use in medical research and applications. Therefore, a need exists for label-free super-resolution techniques, which could be especially valuable for clinical applications. To answer this need, several such techniques have been recently developed. In this chapter, we introduce the reader to some of these techniques, discuss their applications, and of the possible future directions. In order to limit the scope of this vastly expanding topic, we focus on techniques based on structured illumination and intrinsic nonlinear responses of materials.

We investigate optical second-harmonic generation (SHG) from metasurfaces where noncentrosymmetric V-shaped gold nanoparticles are ordered into regular array configurations. In contrast to expectations, a substantial enhancement of the SHG signal is observed when the number density of the particles in the array is reduced. More specifically, by halving the number density, we obtain over five-fold enhancement in SHG intensity. This striking result is attributed to favorable interparticle interactions mediated by the lattice, where surface-lattice resonances lead to spectral narrowing of the plasmon resonances. Importantly, however, the results cannot be explained by the improved quality of the plasmon resonance alone. Instead, the lattice interactions also lead to further enhancement of the local fields at the particles. The experimental observations agree very well with results obtained from numerical simulations including lattice interactions., 17 pages (+10 SI), 4 figures (+7 SI), 1 table

Metal nanoparticles demonstrate unique optical properties that are mostly due to localized surface plasmon resonances (LSPRs). In addition, when nanoparticles are arranged in arrays (metasurfaces), their responses can be modified by the presence of the neighboring particles. As a result, sharp spectral features can be observed. Such features, called surface lattice resonances (SLRs), are related to the appearance of diffraction orders in the optical response. Both types of resonances can lead to local-field enhancement and thereby boost nonlinear optical effects. For the particular case of second-harmonic generation (SHG) the sample needs to be also non-centrosymmetric. This condition is fulfilled when, for example, V-shaped nanoparticles are used in the array. Increasing the number of particles typically increases the optical density, which should increase the nonlinear response with the square of the particle density. This approach, however, has its limitations because, when the particles are too close to each other, the quality of the LSPRs decreases leading to an effect opposite to the desired. Here, we will show the counterintuitive effect that the nonlinear response can be enhanced by reducing the number of particles in the array. In order to verify our idea, we use two arrays of V-shaped gold nanoparticles fabricated on a glass substrate by electron-beam lithography and lift-off methods. The particles are distributed in 500 x 500 nm2 square arrays in two configurations: i) all lattice points are filled with particles (V1) or ii) every other particle in the lattice is removed in a way that the remaining particles form a rotated (by 45°) square array with a pitch of 707 nm (V2). Both samples have two eigenpolarizations: one along the symmetry axis (y) of the V shape and other in the perpendicular direction (x). In the SHG experiments, the incident beam from an optical parametric oscillator was incident on the sample. Polarizers and a half-wave plate were used to control the polarization of the fundamental (1000 – 1300 nm) and second-harmonic beams. The SHG signal was collected by a photon counting system. The sample V2, that has reduced (by a factor of 2) density of particles in the array, shows the expected decrease in the strength of the resonance peak (1151 nm) and a slight redshift of the resonance wavelength with respect to the sample V1 (1081 nm). In order to achieve fair comparison of the nonlinear signals, we tuned the incident wavelength to the position of approximately equal losses for both samples (1135 nm). The sample V2 is found to have, by a factor of 7, stronger response than sample V1. Such enhancement in the nonlinearity is related to the improvement in the quality of the resonance for sample V2, for which the width of the resonance is reduced by ~30% compared to V1. This is due to SLRs that are present for sample V2. Our results are in good agreement with calculations by using an approach based on the discrete-dipole approximation.

The optical properties of metal nanostructures result from their localized surface plasmon resonances (LSPRs). The exact spectral positions of LSPRs depend on the geometry of the particles and optical parameters of the surrounding material. In addition, when the nanoparticles are arranged in an array (metasurface) the LSPRs associated with isolated particles are affected by the presence of other array members. As a result, sharp spectral features related to surface lattice resonances (SLRs) can be observed. SLRs are sensitive to the angle of incidence of incoming light due to their association with the appearance and disappearance of diffraction orders in the optical response. The resonances lead to strong local fields (hot spots) in the proximity of the particles. Such hot spots are particularly important when one wants to enhance nonlinear optical effects, for example second-harmonic generation (SHG). In the case of SHG, the sample needs to be also non-centrosymmetric, which can be fulfilled by using, e.g., V-shaped nanoparticles. In this paper, we show that SHG from arrays of V-shaped gold nanoparticles can be enhanced when the angle of incidence is matching optimal conditions for the excitation of SLRs. Our sample consists of an array of V-shaped nanoparticles fabricated by standard electron-beam lithography and lift-off techniques. The gold particles are distributed in the array with a spacing of 500 nm and have the same dimensions to obtain a resonance close to fundamental wavelength for the polarization along the symmetry axis. The SHG experiments were performed in transmission mode with the incident beam weakly focused on the samples. Polarizers and a half-wave plate were used to control the polarizations of the fundamental and second-harmonic beams. The SHG signal was collected by a photon-counting system for varying incident angles (rotation with respect to the symmetry axis (y) of the sample or orthogonal to it (x)). SLRs can modify LSPRs leading to visible features in the SHG response. When the incident angle is increased, a redshift towards fundamental wavelength is observed together with narrowing of the resonance. Such improvement in the quality of the resonance results in stronger SHG. Thus, the sample needs to be rotated significantly to meet the optimal conditions for SHG enhancement, which can be as high as by a factor of four (projection x-z) or a factor of 10 (projection y-z) comparing to SHG measured at normal incidence. The maximum enhancement of the SHG signal is related to the SLRs which occur near incident angles that allow diffraction orders to propagate along the sample plane (in air or in the substrate). Our investigation of the role of SLRs in SHG from metasurfaces shows that such resonances lead to prominent features in the angle-dependent SHG responses, which results in the enhancement of SHG by a factor of up to ten. In order to achieve the optimal conditions of SHG enhancement the sample needs to be rotated from normal incidence to match the angle that allows diffraction order to propagate in the substrate.

We investigate the role of surface-lattice resonances (SLRs) in second-harmonic generation (SHG) from arrays of metal nanoparticles. The SLRs affect the generated signal when the sample is rotated away from normal incidence. The adjustment of the incident angle tunes the SLRs to the fundamental wavelength for SHG and improves the quality of the resonance for better resonance enhancement of SHG. Compared to normal incidence, an enhancement by a factor of 10 is observed. However, at certain incident angles, the enhancement is interrupted by diffraction anomalies, which redirect light into the substrate, increasing radiation damping and compromising the quality of the resonance.

Highly endurance-trained athlete's heart represents the most extreme form of cardiac adaptation to physical stress, but its circulatory alterations remain obscure. In the present study, myocardial blood flow (MBF), blood mean transit time (MTT), oxygen extraction fraction (OEF) and consumption (MVO2), and efficiency of cardiac work were quantified in highly trained male endurance athletes and control subjects at rest and during supine cycling exercise using [O-15]-labeled radiotracers and positron emission tomography. Heart rate and MBF were lower in athletes both at rest and during exercise. OEF increased in response to exercise in both groups, but was higher in athletes (70 +/- A 21 vs. 63 +/- A 11 % at rest and 86 +/- A 13 vs. 73 +/- A 10 % during exercise). MTT was longer and vascular resistance higher in athletes both at rest and during exercise, but arterial content of 2,3-diphosphoglycerate (oxygen affinity) was unchanged. MVO2 per gram of myocardium trended (p = 0.08) lower in athletes both at rest and during exercise, while myocardial efficiency of work and MVO2 per beat were not different between groups. Arterial levels of free fatty acids were similar to twofold higher in athletes likely leading to higher myocardial fatty acid oxidation and hence oxygen cost, which may have blunted the bradycardia-induced decrease in MVO2. Finally, the observed group differences in MBF, OEF, MTT and vascular resistance remained significant also after they were controlled for differences in MVO2. In conclusion, in highly endurance-trained human heart, increased myocardial blood transition time enables higher oxygen extraction levels with a lower myocardial blood flow and higher vascular resistance. These physiological adaptations to exercise training occur independently of the level of oxygen consumption and together with training-induced bradycardia may serve as mechanisms to increase functional reserve of the human heart.

Autonomic nervous system modifies atrial electrophysiologic properties and arrhythmia vulnerability.Heart rate (HR) variability, an indicator of cardiac autonomic regulation, was measured in 784 subjects (mean age 51 ± 6 years; 54% males) from a standardized 45-minute period in a study population (n = 1,045), which consisted of randomly selected hypertensive and age- and sex-matched control subjects at the time of recruitment in 1991-1992 (the OPERA study).During a mean follow-up of 16.5 ± 3.5 years, 76 subjects (9.7%) had developed symptomatic atrial fibrillation (AF), needing hospitalization. HR did not predict the occurrence of AF. Among the various spectral and time-domain HR variability indexes, only the low-frequency (LF) spectral component independently predicted AF. In the Cox regression analysis, the hazard ratio of reduced HR corrected LF (LFccv ≤ 1.59%, optimal cutoff from the ROC curve) in predicting the AF was 3.28 (95% CI: 2.06-5.24; P0.001). In the multiple Cox regression model, including LFccv and other predictors of AF, such as age, gender, hypertension, history of coronary artery disease, systolic and diastolic blood pressure, body mass index, β-blocking, angiotensin converting enzyme inhibitor and aspirin medication, left atrial size, left ventricular mass index, and left ventricular size obtained by echocardiography, only LFccv (hazard ratio 2.81; 95% CI: 1.64-4.81; P0.001), age (P = 0.006), and systolic blood pressure (P = 0.02) remained as significant predictors of AF.Impaired LF oscillation of HR predicts new-onset AF in a middle-aged population emphasizing the important role of autonomic nervous system in the genesis of symptomatic AF.

Background: QRS/T angle and the cosine of the angle between QRS and T‐wave vectors (TCRT), measured from standard 12‐lead electrocardiogram (ECG), have been used in risk stratification of patients. This study assessed the possible rate dependence of these variables during exercise ECG in healthy subjects. Methods: Forty healthy volunteers, 20 men and 20 women, aged 34.6 ± 3.4, underwent an exercise ECG testing. Twelve‐lead ECG was recorded from each test subject and the spatial QRS/T angle and TCRT were automatically analyzed in a beat‐to‐beat manner with custom‐made software. The individual TCRT/RR and QRST/RR patterns were fitted with seven different regression models, including a linear model and six nonlinear models. Results: TCRT and QRS/T angle showed a significant rate dependence, with decreased values at higher heart rates (HR). In individual subjects, the second‐degree polynomic model was the best regression model for TCRT/RR and QRST/RR slopes. It provided the best fit for both exercise and recovery. The overall TCRT/RR and QRST/RR slopes were similar between men and women during exercise and recovery. However, women had predominantly higher TCRT and QRS/T values. With respect to time, the dynamics of TCRT differed significantly between men and women; with a steeper exercise slope in women (women, −0.04/min vs −0.02/min in men, P < 0.0001). In addition, evident hysteresis was observed in the TCRT/RR slopes; with higher TCRT values during exercise. Conclusions: The individual patterns of TCRT and QRS/T angle are affected by HR and gender. Delayed rate adaptation creates hysteresis in the TCRT/RR slopes. Ann Noninvasive Electrocardiol 2010;15(3):264–275

Background. Changes in cardiac autonomic function have been documented after myocardial infraction (MI), but their significance is unknown. We assessed the amount of recovery of autonomic dysfunction after MI and its significance among the patients with depressed left ventricular ejection fraction (LVEF ≤ 0.40 - CARISMA and ≤ 0.50 - REFINE). Methods and Results. Changes in heart rate variability (dHRV) and turbulence (dHRT) were measured from 24-hour ECG recordings performed 5–21 days and again at six weeks post-MI in 312 patients included in the Cardiac Arrhythmias and Risk Stratification after Myocardial Infarction (CARISMA) study. The primary endpoint was sustained ventricular tachycardia (VT) or ventricular fibrillation (VF) recorded by an implantable device. Non-arrhythmic death was a secondary endpoint. HRV and HRT measures increased significantly from baseline to six weeks post-AMI (p Conclusions. Autonomic function undergoes a substantial improvement early after MI, which is associated with vulnerability to VT or VF. A marked recovery of autonomic dysfunction protects from the occurrence of fatal or near-fatal arrhythmic events after MI despite depressed LVEF

The optical properties of metal nanoparticles are mostly due to localized surface plasmon resonances. Such resonances can be strongly modified by interparticle coupling when the particles are arranged in regular arrays. We investigate the role of lattice effects in the second-harmonic generation from arrays of metal nanoparticles.