National Enterprise for NanoScience and NanoTechnology

Background The integration of graphene in photovoltaic modules, fuel cells, batteries, supercapacitors, and devices for hydrogen generation offers opportunities to tackle challenges driven by the increasing global energy demand. Graphene’s two-dimensional (2D) nature leads to a theoretical surface-to-mass ratio of ~2600 m 2 /g, which combined with its high electrical conductivity and flexibility, gives it the potential to store electric charge, ions, or hydrogen. Other 2D crystals, such as transition metal chalcogenides (TMDs) and transition metal oxides, are also promising and are now gaining increasing attention for energy applications. The advantage of using such 2D crystals is linked to the possibility of creating and designing layered artificial structures with “on-demand” properties by means of spin-on processes, or layer-by-layer assembly. This approach exploits the availability of materials with metallic, semiconducting, and insulating properties. Advances The success of graphene and related materials (GRMs) for energy applications crucially depends on the development and optimization of production methods. High-volume liquid-phase exfoliation is being developed for a wide variety of layered materials. This technique is being optimized to control the flake size and to increase the edge-to-surface ratio, which is crucial for optimizing electrode performance in fuel cells and batteries. Micro- or nanocrystal or flake edge control can also be achieved through chemical synthesis. This is an ideal route for functionalization, in order to improve storage capacity. Large-area growth via chemical vapor deposition (CVD) has been demonstrated, producing material with high structural and electronic quality for the preparation of transparent conducting electrodes for displays and touch-screens, and is being evaluated for photovoltaic applications. CVD growth of other multicomponent layered materials is less mature and needs further development. Although many transfer techniques have been developed successfully, further improvement of high-volume manufacturing and transfer processes for multilayered heterostructures is needed. In this context, layer-by-layer assembly may enable the realization of devices with on-demand properties for targeted applications, such as photovoltaic devices in which photon absorption in TMDs is combined with charge transport in graphene. Outlook Substantial progress has been made on the preparation of GRMs at the laboratory level. However, cost-effective production of GRMs on an industrial scale is needed to create the future energy value chain. Applications that could benefit the most from GRMs include flexible electronics, batteries with efficient anodes and cathodes, supercapacitors with high energy density, and solar cells. The realization of GRMs with specific transport and insulating properties on demand is an important goal. Additional energy applications of GRMs comprise water splitting and hydrogen production. As an example, the edges of MoS 2 single layers can oxidize fuels—such as hydrogen, methanol, and ethanol—in fuel cells, and GRM membranes can be used in fuel cells to improve proton exchange. Functionalized graphene can be exploited for water splitting and hydrogen production. Flexible and wearable devices and membranes incorporating GRMs can also generate electricity from motion, as well as from water and gas flows. Tailored GRMs for energy applications. The ability to produce GRMs with desired specific properties paves the way to their integration in a variety of energy devices. Solution processing and chemical vapor deposition are the ideal means to produce thin films that can be used as electrodes in energy devices (such as solar panels, batteries, fuel cells, or in hydrogen storage). Chemical synthesis is an attractive route to produce “active” elements in solar cell or thermoelectric devices.

Quantum mechanics, through the Heisenberg uncertainty principle, imposes limits on the precision of measurement. Conventional measurement techniques typically fail to reach these limits. Conventional bounds to the precision of measurements such as the shot noise limit or the standard quantum limit are not as fundamental as the Heisenberg limits and can be beaten using quantum strategies that employ "quantum tricks" such as squeezing and entanglement.

Graphene hosts a unique electron system in which electron-phonon scattering is extremely weak but electron-electron collisions are sufficiently frequent to provide local equilibrium above the temperature of liquid nitrogen. Under these conditions, electrons can behave as a viscous liquid and exhibit hydrodynamic phenomena similar to classical liquids. Here we report strong evidence for this transport regime. We found that doped graphene exhibits an anomalous (negative) voltage drop near current-injection contacts, which is attributed to the formation of submicrometer-size whirlpools in the electron flow. The viscosity of graphene's electron liquid is found to be ~0.1 square meters per second, an order of magnitude higher than that of honey, in agreement with many-body theory. Our work demonstrates the possibility of studying electron hydrodynamics using high-quality graphene.

Hydrogen-based fuel cells are promising solutions for the efficient and clean delivery of electricity. Since hydrogen is an energy carrier, a key step for the development of a reliable hydrogen-based technology requires solving the issue of storage and transport of hydrogen. Several proposals based on the design of advanced materials such as metal hydrides and carbon structures have been made to overcome the limitations of the conventional solution of compressing or liquefying hydrogen in tanks. Nevertheless none of these systems are currently offering the required performances in terms of hydrogen storage capacity and control of adsorption/desorption processes. Therefore the problem of hydrogen storage remains so far unsolved and it continues to represent a significant bottleneck to the advancement and proliferation of fuel cell and hydrogen technologies. Recently, however, several studies on graphene, the one-atom-thick membrane of carbon atoms packed in a honeycomb lattice, have highlighted the potentialities of this material for hydrogen storage and raise new hopes for the development of an efficient solid-state hydrogen storage device. Here we review on-going efforts and studies on functionalized and nanostructured graphene for hydrogen storage and suggest possible developments for efficient storage/release of hydrogen under ambient conditions.

An interesting observation, reported for transgenic plants that have been engineered to overproduce osmolytes, is that they often exhibit impaired growth in the absence of stress. As growth reduction and accumulation of osmolytes both typically result from adaptation, we hypothesized that growth reduction may actually result from osmolyte accumulation. To examine this possibility more closely, intracellular proline level was manipulated by expressing mutated derivatives of tomPRO2 (a Delta(1)-pyrroline-5-carboxylate synthetase, P5CS, from tomato) in Saccharomyces cerevisiae. This was done in the presence and absence of a functional proline oxidase, followed by selection and screening for increased accumulation of proline in the absence of any stress. Here we show, in support of our hypothesis, that the level of proline accumulation and the amount of growth are inversely correlated in cells grown under normal osmotic conditions. In addition, the intracellular concentration of proline also resulted in increases in ploidy level, vacuolation and altered accumulation of several different transcripts related to cell division and gene expression control. Because these cellular modifications are common responses to salt stress in both yeast and plants, we propose that proline and other osmolytes may act as a signaling/regulatory molecule able to activate multiple responses that are part of the adaptation process. As in previous studies with transgenic plants that overaccumulate osmolytes, we observed some increase in relative growth of proline-overaccumulating cells in mild hyperosmotic stress.

A hallmark of graphene is its unusual conical band structure that leads to a zero-energy band gap at a single Dirac crossing point. By measuring the spectral function of charge carriers in quasi-freestanding graphene with angle-resolved photoemission spectroscopy, we showed that at finite doping, this well-known linear Dirac spectrum does not provide a full description of the charge-carrying excitations. We observed composite "plasmaron" particles, which are bound states of charge carriers with plasmons, the density oscillations of the graphene electron gas. The Dirac crossing point is resolved into three crossings: the first between pure charge bands, the second between pure plasmaron bands, and the third a ring-shaped crossing between charge and plasmaron bands.

Using data from a credit card issuer, a neural network based fraud detection system was trained on a large sample of labelled credit card account transactions and tested on a holdout data set that consisted of all account activity over a subsequent two-month period of time. The neural network was trained on examples of fraud due to lost cards, stolen cards, application fraud, counterfeit fraud, mail-order fraud and NRI (non-received issue) fraud. The network detected significantly more fraud accounts (an order of magnitude more) with significantly fewer false positives (reduced by a factor of 20) over rule-based fraud detection procedures. We discuss the performance of the network on this data set in terms of detection accuracy and earliness of fraud detection. The system has been installed on an IBM 3090 at Mellon Bank and is currently in use for fraud detection on that bank's credit card portfolio.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Since its inception about two centuries ago thermodynamics has sparkled continuous interest and fundamental questions. According to the second law no heat engine can have an efficiency larger than Carnot's efficiency. The latter can be achieved by the Carnot engine, which however ideally operates in infinite time, hence delivers null power. A currently open question is whether the Carnot efficiency can be achieved at finite power. Most of the previous works addressed this question within the Onsager matrix formalism of linear response theory. Here we pursue a different route based on finite-size-scaling theory. We focus on quantum Otto engines and show that when the working substance is at the verge of a second order phase transition diverging energy fluctuations can enable approaching the Carnot point without sacrificing power. The rate of such approach is dictated by the critical indices, thus showing the universal character of our analysis.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of extracellular amyloid-beta peptide (Aβ) and intracellular neurofibrillar tangles, associated with loss of neurons in the brain and consequent learning and memory deficits. Aβ is the major component of the senile plaques and is believed to play a central role in the development and progress of AD both in oligomer and fibril forms. Inhibition of the formation of Aβ fibrils as well as the destabilization of preformed Aβ in the Central Nervous System (CNS) would be an attractive therapeutic target for the treatment of AD. Moreover, a large number of studies indicate that oxidative stress and mitochondrial dysfunction may play an important role in AD and their suppression or reduction via antioxidant use could be a promising preventive or therapeutic intervention for AD patients. Many antioxidant compounds have been demonstrated to protect the brain from Aβ neurotoxicity. Ferulic acid (FA) is an antioxidant naturally present in plant cell walls with anti-inflammatory activities and it is able to act as a free radical scavenger. Here we present the role of FA as inhibitor or disaggregating agent of amyloid structures as well as its effects on biological models.

Seizures in focal epilepsies are sustained by a highly synchronous neuronal discharge that arises at restricted brain sites and subsequently spreads to large portions of the brain. Despite intense experimental research in this field, the earlier cellular events that initiate and sustain a focal seizure are still not well defined. Their identification is central to understand the pathophysiology of focal epilepsies and to develop new pharmacological therapies for drug-resistant forms of epilepsy. The prominent involvement of astrocytes in ictogenesis was recently proposed. We test here whether a cooperation between astrocytes and neurons is a prerequisite to support ictal (seizure-like) and interictal epileptiform events. Simultaneous patch-clamp recording and Ca2+ imaging techniques were performed in a new in vitro model of focal seizures induced by local applications of N-methyl-D-aspartic acid (NMDA) in rat entorhinal cortex slices. We found that a Ca2+ elevation in astrocytes correlates with both the initial development and the maintenance of a focal, seizure-like discharge. A delayed astrocyte activation during ictal discharges was also observed in other models (including the whole in vitro isolated guinea pig brain) in which the site of generation of seizure activity cannot be precisely monitored. In contrast, interictal discharges were not associated with Ca2+ changes in astrocytes. Selective inhibition or stimulation of astrocyte Ca2+ signalling blocked or enhanced, respectively, ictal discharges, but did not affect interictal discharge generation. Our data reveal that neurons engage astrocytes in a recurrent excitatory loop (possibly involving gliotransmission) that promotes seizure ignition and sustains the ictal discharge. This neuron-astrocyte interaction may represent a novel target to develop effective therapeutic strategies to control seizures.

The combination of materials with targeted optical properties and of complex, 3D architectures, which can be nowadays obtained by additive manufacturing, opens unprecedented opportunities for developing new integrated systems in photonics and optoelectronics. The recent progress in additive technologies for processing optical materials is here presented, with emphasis on accessible geometries, achievable spatial resolution, and requirements for printable optical materials. Relevant examples of photonic and optoelectronic devices fabricated by 3D printing are shown, which include light-emitting diodes, lasers, waveguides, optical sensors, photonic crystals and metamaterials, and micro-optical components. The potential of additive manufacturing applied to photonics and optoelectronics is enormous, and the field is still in its infancy. Future directions for research include the development of fully printable optical and architected materials, of effective and versatile platforms for multimaterial processing, and of high-throughput 3D printing technologies that can concomitantly reach high resolution and large working volumes.

Artificial crystal lattices can be used to tune repulsive Coulomb interactions between electrons. We trapped electrons, confined as a two-dimensional gas in a gallium arsenide quantum well, in a nanofabricated lattice with honeycomb geometry. We probed the excitation spectrum in a magnetic field, identifying collective modes that emerged from the Coulomb interaction in the artificial lattice, as predicted by the Mott-Hubbard model. These observations allow us to determine the Hubbard gap and suggest the existence of a Coulomb-driven ground state.

Exciton bound states in solids between electrons and holes are predicted to form a superfluid at high temperatures. We show that by employing atomically thin crystals such as a pair of adjacent bilayer graphene sheets, equilibrium superfluidity of electron-hole pairs should be achievable for the first time. The transition temperatures are well above liquid helium temperatures. Because the sample parameters needed for the device have already been attained in similar graphene devices, our work suggests a new route toward realizing high-temperature superfluidity in existing quality graphene samples.

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.

Diamond-like carbon (DLC)-coated cotton textiles showing both superhydrophobic and superoleophilic properties exhibit highly controllable, energy-efficient oil–water separation.

Iron oxide nanoparticles (NPs) are frequently employed in biomedical research as magnetic resonance (MR) contrast agents where high intracellular levels are required to clearly depict signal alterations. To date, the toxicity and applicability of these particles have not been completely unraveled. Here, we show that endosomal localization of different iron oxide particles results in their degradation and in reduced MR contrast, the rate of which is governed mainly by the stability of the coating. The release of ferric iron generates reactive species, which greatly affect cell functionality. Lipid-coated NPs display the highest stability and furthermore exhibit intracellular clustering, which significantly enhances their MR properties and intracellular persistence. These findings are of considerable importance because, depending on the nature of the coating, particles can be rapidly degraded, thus completely annihilating their MR contrast to levels not detectable when compared to controls and greatly impeding cell functionality, thereby hindering their application in functional in vivo studies.

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.

Dendrimers have been described as one of the most tunable and therefore potentially applicable nanoparticles both for diagnostics and therapy. Recently, in order to realize drug delivery agents, most of the effort has been dedicated to the development of dendrimers that could internalize into the cells and target specific intracellular compartments in vitro and in vivo. Here, we describe cell internalization properties and diffusion of G4 and G4-C12 modified PAMAM dendrimers in primary neuronal cultures and in the CNS of live animals. Confocal imaging on primary neurons reveals that dendrimers are able to cross the cell membrane and reach intracellular localization following endocytosis. Moreover, functionalization of PAMAMs has a dramatic effect on their ability to diffuse in the CNS tissue in vivo and penetrate living neurons as shown by intraparenchymal or intraventricular injections. 100 nM G4-C12 PAMAM dendrimer already induces dramatic apoptotic cell death of neurons in vitro. On the contrary, G4 PAMAM does not induce apoptotic cell death of neural cells in the sub-micromolar range of concentration and induces low microglia activation in brain tissue after a week. Our detailed description of dendrimer distribution patterns in the CNS will facilitate the design of tailored nanomaterials in light of future clinical applications.

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. A fundamental limitation within the field of digital microfluidics is the minimum droplet volume that can be internally-controlled and exploited. Surface acoustic waves (SAWs) have been shown to be a fast and efficient method for generating internal flows and patterning in microfluidic droplets, but no improvements in droplet miniaturization have been reported since its introduction. Here we demonstrate the relevant length scales in sub-nanometer amplitude SAW-driven acoustic streaming. We illustrate the absence of any physical limitations beyond fabrication capabilities preventing the downscaling of SAW-driven internal streaming to nanoliter microreactors and beyond, and we experimentally demonstrate this by extending SAW microfluidics up to operating frequencies in the GHz range. The scaling is applied to demonstrate ultrafast fluid mixing in nanoliter order droplets by exploiting 1.1 GHz SAW. Development of micro-total-analysis systems offers new possibilities in portable healthcare and environmental sensor systems. SAW driven microfluidics has become a very powerful fluid actuation method owing to the ability of SAWs to transfer a large amount of momentum into fluids, using integrated transducers in truly portable battery-operated systems.1 The typical amplitudes of SAWs used in these devices are on the order of nanometers or less, however owing to the high frequencies used (O(10 − 100 MHz)) the accelerations induced by these waves are enormous—over 107 ms−2—which can be exploited to drive ultra-fast flows in microfluids.2 Depending on geometry and power input, SAWs can actuate a range of fluid processes such as mixing,3, 4 particle manipulation,5-7 droplet actuation,8-10 atomization,11 water-in-oil emulsions,12 and pumping in closed microchannels via acoustic counterflow.13, 14 Digital SAW microfluidics applications have typically been demonstrated using microliter droplets or larger, as in the case of other digital microfluidics technologies.15 Further miniaturization beyond these limits, including fine control of internal flows in nanoliter order microreactors, is imperative to address as we look to the future of digital microfluidics. The outstanding performance of SAW-driven micro-mixers, micro-centrifuges and micro-sorters5 arises from SAW-driven acoustic streaming. SAWs radiate acoustic energy into fluids owing to the sound velocity mismatch between the fluid and the substrate. The induced pressure wave in turn drives a steady state flow, namely acoustic streaming. Up to now SAW microfluidic devices have typically been operated with frequencies between 20 MHz to 200 MHz, with acoustic wavelengths above ∼20 μm (although they have been exploited very recently up to ∼300 MHz16, 17). No scaling laws of the resulting flow patterns as a function of the acoustic length scales has been explicitly investigated. In order to downscale SAW microfluidics, we have studied for the first time here acoustic streaming and particle patterning as a function of the operating SAW frequency from 47.8 − 1107 MHz while varying the droplet volumes from microliters down to <∼ 1 nanoliter. We demonstrate how acoustic streaming and particle patterning scales with the ratio of droplet sizes to viscous fluid acoustic damping and SAW damping lengths. For the first time acoustic streaming and ultrafast mixing in nanoliter order droplets is investigated by exploiting SAWs in the ultra high frequency range (UHF; defined as 300 MHz – 3 GHz). SAW microfluidic devices, as shown in Figure 1, were fabricated at nominal frequencies of 50, 100, 200, 400, 833, and 1250 MHz, and the reflection coefficients were measured to find the resonant frequency at which to operate (Table 1; details of similar fabrication techniques can be found in Travagliati et al.18). Each SAW device consisted of straight-fingered titanium:gold (10 nm:100 nm) interdigital transducers (IDT) patterned on 128° Y-cut, X-propagating lithium niobate (LN) substrates. For the purpose of microfluidic actuation, the amplitude of the SAWs generated by each device was set to A = 100 pm by direct measurement with a laser Doppler vibrometer (LDV; UHF-120 Ultra High Frequency Vibrometer, Polytec, Germany). The SAW amplitude was measured in the region where the fluid was placed, and was found to be consistent across the wave path. To visualize internal streaming flows, droplets were pinned in hydrophilic well regions formed by patterned surface silanes. The contact angle for each droplet on the surface was 93° ± 3°. For the case of the large droplets in Figure 2 only, a coverslip was placed over the droplets with 385 μm spacers to aid in flow visualization. Similar setups were used to demonstrate microfluidic mixing, with initially segregated fluid sections. For 1 μL droplets 10% dye was initially compartmentalized in water, and for the nanoliter scale droplets latex beads (500 nm) were used. Mixing efficiency was determined by a normalized mixing index over time, with an associated mixing half-life (details in Ref. [4]). Figure 2 shows the flow streamlines (from overlaid images of particle tracers) of representative measurements for the different operating frequencies used to actuate internal streaming in 1 μL particle seeded water droplets of diameter d = 1.5 mm. A frequency dependent transition between two distinct flow patterns was seen as the frequency was increased. Streamlines with standard double vortical structures19 were observed for the droplets actuated by resonant frequencies up to 374 MHz. While this flow structure was apparent at frequencies that are typically associated with SAW microfluidics, at frequencies above 374 MHz a different flow structure appeared. This new flow pattern was characterized by two regions; one at the leading edge and one at the rear of the droplet. In the front region, again, there was a symmetrical vortical pattern with a strong three dimensional (3D) structure in which particles move upwards away from the SAW scattering region. The back region instead was characterized by a single rolling motion. As the frequency was increased from 752 MHz to 1.107 GHz the front vortices were compressed further and the back-rolling motion had the appearance of becoming increasingly chaotic. The flow pattern transition can be described in terms of the decreased damping length of the acoustic wave in a viscous fluid, xf, as the frequency was increased. The damping length of an acoustic wave in water is given by xf = 1/(γ kf), where kf = ω/cf and γ is (1+β)ηω/(2ρf cf2), where γ is the non-dimensional acoustic damping factor, cf is the speed of sound in fluid, β is the non-dimensional viscosity ratio (5/3 for simple fluids such as water), and η is the kinematic viscosity20—given for each frequency in Table 1. The standard vortex pairs were generated by the 47.9 − 374 MHz devices, where xf was greater (or on the order of) the droplet height h. For the devices with frequencies above 374 MHz, where there was the new strongly 3D fluid flow, the acoustic wave in the fluid died out at a decreasing fraction of the droplet size. The standard dual vortex flows can be expected for cases where h/xf < 1 and the new compartmentalized 3D pattern will occur in cases of h/xf > 1. Where h < xf, a significant part of the acoustic energy was reflected at the surface of the droplet, generating a pressure field over the droplet length which induced a jet extension (central high velocity fluid path) over the entire droplet as in the standard pattern. Conversely, where h > xf the acoustic wave energy was dissipated before reflection at droplet surface and a strong gradient was defined only in the front region of the droplet. We in turn saw the jet and vortex pairs (the region of most intense fluid velocities) withdrawing closer to the fluid front—a change from the two-dimensional flows of lower frequency devices previously studied. Here, the pressure wave was compressed in the front region and its gradient was stronger in the out-of-plane direction. In the rear of the droplets where there was the single-rolling motion, instead, the pressure field had almost vanished. As the frequency was increased from 752 MHz to 1.107 GHz the back-rolling motion had the appearance of becoming increasingly chaotic. This may be expected as the acoustic intensity radiated into the droplet scales as I ∼ f 2A2 and it has been shown that at a fixed low frequency an increase of acoustic power leads to an increase of chaos in the flow structure.4, 5 Figure 3 demonstrates downscaling of the SAW microfluidic technology (Video S1). Flow patterns inside three significantly smaller nanoliter order droplets actuated by four frequencies are shown; (a) 47.8 MHz, (b) 94.9 MHz, (c) 191 MHz and (d) 1107 MHz (water seeded with 500 nm particles; 374 MHz and 752 MHz are not shown as they were qualitatively similar to 1107 MHz). For each droplet in the 47.8 MHz case (Figure 3(a)), there was a departure from the vortical structure seen in Figure 2 and particles accumulated in rings as standing waves were formed in the droplet, reminiscent of Li et al.’s pattern generation in larger droplets by low (20 MHz) frequency SAW.21 For the 94.9 MHz and 191 MHz induced flows there was a transition from the vortical structure seen in the larger droplet (and in Figure 2) to ring-patterns of the particles in the smaller droplets. The 94.9 MHz driven flows in the 26 nL droplet (Figure 3(b) left) and the 191 MHz driven flows in the 6.6 nL droplet (Figure 3(c) center) both exhibited transitional behaviors, displaying a combination of dual vortices and standing waves. For the 1107 MHz case (Figure 3(d)), the standard pair of vortices formed again in each case, demonstrating acoustic streaming flow in the 1.2 nL droplet. The smallest droplets in Figure 3(a–c) also showed that the distance between the standing wave driven accumulation lines decreased with the decrease in acoustic wavelength (increase in operating frequency). By adjusting the frequency of operation within this range, particle positioning and patterning could be controlled. This streaming-to-particle accumulation transition above can be described in terms of the droplet size d compared to the SAW damping length, xs = 0.45 λs (ρscs)/(ρf cf) where λs, ρs and cs are the wavelength, density and speed of the SAW in the substrate respectively.22 Acoustic streaming is generated when d/xs > 1, while particle accumulation lines occur when d/xs ≪ 1. At the transition d/xs ∼ 1, particle dynamics resulting in a combination of the two cases occurs. When xs ≪ d the acoustic source at the substrate-fluid interface has a substantial gradient, typical of previous SAW digital microfluidics investigations. If, however, xs > d, the acoustic field in the substrate at the solid-liquid interface has minimal decay over the droplet length resulting in an acoustofluidics behavior similar to bulk acoustic wave (BAW) actuated devices where standing waves appear in the fluid. In this situation particle accumulation along anti-nodal lines dominates over the acoustic streaming as has been theoretically shown for fluid reactor geometries with aspect ratios of less than two.23, 24 We now exploit the UHF-SAW driven streaming results from the previous sections to demonstrate one of the most important, difficult and limiting aspects of microfluidics—efficient and fast fluid mixing in microdroplets. Figure 4(a) shows the mixing half-lives for 1 μL droplets over the investigated range of frequencies (as in Figure 2). We find that fast mixing is obtained for all the tested frequencies. Mixing times (Mτ) are reduced by increasing the SAW frequency while keeping the SAW amplitude (A = 100 pm) constant, and scales as Mτ ∼ f−2. This is a convenient scaling law and shows that Mτ scales with the inverse of the acoustic intensity, I, with a slight deviation at the highest frequency, possibly owing to some of the energy being used to generate droplet atomization.11 The truly impacting application, however, is shown in Figure 4(b)–(d), where we apply the 1.1 GHz generated streaming to drive mixing in nanoliter order droplets. As shown in Figure 3, standing wave patterns and particle accumulations are generated in nanoliter scale droplets at typical SAW frequencies, which are not beneficial for mixing. Here, we instead exploit the UHF regime of acoustic streaming to generate mixing flows in these very small droplets. Figure 4(b) and (c) shows typical comparison images over time between mixing in a ∼6 nL droplet via (b) diffusion alone, and (c) 1.1 GHz SAW driven acoustic streaming. We see a dramatic decrease in mixing time of over two orders of magnitude. In conclusion, we demonstrated that the necessary condition for vortical streaming in nanoliter volume droplets is xs ≪ d, where there is a sufficient pressure gradient to initiate acoustic streaming rather than standing wave patterns. Additionally, as xf becomes significantly small with respect to the droplet dia­meter the vortical patterns compress to the leading edge and the bulk flow becomes increasingly chaotic and three dimensional. By increasing the frequency of operation we can restore the standard SAW patterns that we see at lower, common operating frequencies, but now in significantly smaller droplets—microliters can in this way be reduced to nanoliters. By accessing frequencies up to the GHz range as shown here, tailored internal flow patterns can be induced in much smaller fluid droplets than were previously available by other microfluidic methods. This was applied to demonstrate a reduction of over two orders of magnitude in mixing time in nanoliter order droplets as compared to diffusion alone. Acoustic streaming patterns and mixing in nanoliter free-droplets are shown, which can be exploited for nano-fluidic mixers and particle manipulations, and extended to new types of flows and pattern generations in micro-channels for lab-on-a-chip devices. This work has been supported in part by the CNR project: NANOMAX “Nanotechnology-based therapy and diagnostics of brain diseases – NANOBRAIN”. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract MALDI mass spectrometry imaging is able to simultaneously determine the spatial distribution of hundreds of molecules directly from tissue sections, without labeling and without prior knowledge. Ultra-high mass resolution measurements based on Fourier-transform mass spectrometry have been utilized to resolve isobaric lipids, metabolites and tryptic peptides. Here we demonstrate the potential of 15T MALDI-FTICR MSI for molecular pathology in a mouse model of high-grade glioma. The high mass accuracy and resolving power of high field FTICR MSI enabled tumor specific proteoforms, and tumor-specific proteins with overlapping and isobaric isotopic distributions to be clearly resolved. The protein ions detected by MALDI MSI were assigned to proteins identified by region-specific microproteomics (0.8 mm 2 regions isolated using laser capture microdissection) on the basis of exact mass and isotopic distribution. These label free quantitative experiments also confirmed the protein expression changes observed by MALDI MSI and revealed changes in key metabolic proteins, which were supported by in-situ metabolite MALDI MSI.