Photocell-Based Optofluidic Device for Clogging-Free Cell Transit Time Measurements.

Measuring the transit time of a cell forced through a bottleneck is one of the most widely used techniques for the study of cell deformability in flow. It in turn provides an accessible and rapid way of obtaining crucial information regarding cell physiology. Many techniques are currently being investigated to reliably retrieve this time, but their translation to diagnostic-oriented devices is often hampered by their complexity, lack of robustness, and the bulky external equipment required. Herein, we demonstrate the benefits of coupling microfluidics with an optical method, like photocells, to measure the transit time. We exploit the femtosecond laser irradiation followed by chemical etching (FLICE) fabrication technique to build a monolithic 3D device capable of detecting cells flowing through a 3D non-deformable constriction which is fully buried in a fused silica substrate. We validated our chip by measuring the transit times of pristine breast cancer cells (MCF-7) and MCF-7 cells treated with Latrunculin A, a drug typically used to increase their deformability. A difference in transit times can be assessed without the need for complex external instrumentation and/or demanding computational efforts. The high throughput (4000-10,000 cells/min), ease of use, and clogging-free operation of our device bring this approach much closer to real scenarios.

Nanoactuator for Neuronal Optoporation.

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing "on-demand" light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation.

Hydrogen-Assisted Thermal Treatment of Electrode Materials for Electrochemical Double-Layer Capacitors.

The capacitance of electrode materials used in electrochemical double-layer capacitors (EDLCs) is currently limited by several factors, including inaccessible isolated micropores in high-surface area carbons, the finite density of states resulting in a quantum capacitance in series to Helmholtz double-layer capacitance, and the presence of surface impurities, such as functional groups and adsorbed species. To unlock the full potential of EDLC active materials and corresponding electrodes, several post-production treatments are commonly proposed to improve their capacitance and, thus, the energy density of the corresponding devices. In this work, we report a systematic study of the effect of a prototypical treatment, namely H2-assisted thermal treatment, on the chemical, structural, and thermal properties of activated carbon and corresponding electrodes. By combining multiple characterization techniques, we clarify the actual origins of the improvement of the performance (e.g., > +35% energy density for the investigated power densities in the 0.5-45 kW kg-1 range) of the EDLCs based on treated electrodes compared to the case based on the pristine electrodes. Contrary to previous works supporting a questionable graphitization of the activated carbon at temperatures <1000 °C, we found that a "surface graphitization" of the activated carbon, detected by spectroscopic analysis, is mainly associated with the desorption of surface contaminants. The elimination of surface impurities, including adsorbed species, improves the surface capacitance of the activated carbon (CsurfAC) by +37.1 and +36.3% at specific currents of 1 and 10 A g-1, respectively. Despite the presence of slight densification of the activated carbon upon the thermal treatment, the latter still improves the cell gravimetric capacitance normalized on the mass of the activated carbon only (CgAC), e.g., + 28% at 1 A g-1. Besides, our holistic approach identifies the change in the active material and binder contents as a concomitant cause of the increase of cell gravimetric capacitance (Cg), accounting for the mass of all of the electrode materials measured for treated electrodes compared to pristine ones. Overall, this study provides new insights into the relationship between the modifications of the electrode materials induced by H2-assisted thermal treatments and the performance of the resulting EDLCs.

Influence of structural colour on the photoprotective mechanism in the gametophyte phase of the red alga Chondrus crispus.

Marine life is populated by a huge diversity of organisms with an incredible range of colour. While structural colour mechanisms and functions are usually well studied in marine animal species, there is a huge knowledge gap regarding the marine macroalgae (red, green and brown seaweeds) that have structural coloration and the biological significance of this phenomenon in these photosynthetic organisms. Here we show that structural colour in the gametophyte life history phase of the red alga Chondrus crispus plays an important role as a photoprotective mechanism in synergy with the other pigments present. In particular, we have demonstrated that blue structural coloration attenuates the more energetic light while simultaneously favouring green and red light harvesting through the external antennae (phycobilisomes) which possess an intensity-dependent photoprotection mechanism. These insights into the relationship between structural colour and photosynthetic light management further our understanding of the mechanisms involved.

The light-dependent pseudo-capacitive charging of conjugated polymer nanoparticles coupled with the depolarization of the neuronal membrane.

The mechanism underlying visual restoration in blind animal models of retinitis pigmentosa using a liquid retina prosthesis based on semiconductive polymeric nanoparticles is still being debated. Through the application of mathematical models and specific experiments, we developed a coherent understanding of abiotic/biotic coupling, capturing the essential mechanism of photostimulation responsible for nanoparticle-induced retina activation. Our modeling is based on the solution of drift-diffusion and Poisson-Nernst-Planck models in the multi-physics neuron-cleft-nanoparticle-extracellular space domain, accounting for the electro-chemical motion of all the relevant species following photoexcitation. Modeling was coupled with electron microscopy to estimate the size of the neuron-nanoparticle cleft and electrophysiology on retina explants acutely or chronically injected with nanoparticles. Overall, we present a consistent picture of electrostatic depolarization of the bipolar cell driven by the pseudo-capacitive charging of the nanoparticle. We demonstrate that the highly resistive cleft composition, due to filling by adhesion/extracellular matrix proteins, is a crucial ingredient for establishing functional electrostatic coupling. Additionally, we show that the photo-chemical generation of reactive oxygen species (ROS) becomes relevant only at very high light intensities, far exceeding the physiological ones, in agreement with the lack of phototoxicity shown in vivo.

Skeletal muscle cells opto-stimulation by intramembrane molecular transducers.

Optical stimulation and control of muscle cell contraction opens up a number of interesting applications in hybrid robotic and medicine. Here we show that recently designed molecular phototransducer can be used to stimulate C2C12 skeletal muscle cells, properly grown to exhibit collective behaviour. C2C12 is a skeletal muscle cell line that does not require animal sacrifice Furthermore, it is an ideal cell model for evaluating the phototransducer pacing ability due to its negligible spontaneous activity. We study the stimulation process and analyse the distribution of responses in multinuclear cells, in particular looking at the consistency between stimulus and contraction. Contractions are detected by using an imaging software for object recognition. We find a deterministic response to light stimuli, yet with a certain distribution of erratic behaviour that is quantified and correlated to light intensity or stimulation frequency. Finally, we compare our optical stimulation with electrical stimulation showing advantages of the optical approach, like the reduced cell stress.

Optofluidic Flow Cytometer with In-Plane Spherical Mirror for Signal Enhancement.

Statistical analysis of the properties of single microparticles, such as cells, bacteria or plastic slivers, has attracted increasing interest in recent years. In this regard, field flow cytometry is considered the gold standard technique, but commercially available instruments are bulky, expensive, and not suitable for use in point-of-care (PoC) testing. Microfluidic flow cytometers, on the other hand, are small, cheap and can be used for on-site analyses. However, in order to detect small particles, they require complex geometries and the aid of external optical components. To overcome these limitations, here, we present an opto-fluidic flow cytometer with an integrated 3D in-plane spherical mirror for enhanced optical signal collection. As a result, the signal-to-noise ratio is increased by a factor of six, enabling the detection of particle sizes down to 1.5 µm. The proposed optofluidic detection scheme enables the simultaneous collection of particle fluorescence and scattering using a single optical fiber, which is crucial to easily distinguishing particle populations with different optical properties. The devices have been fully characterized using fluorescent polystyrene beads of different sizes. As a proof of concept for potential real-world applications, signals from fluorescent HEK cells and Escherichia coli bacteria were analyzed.

Modulation of Mechanosensitive Potassium Channels by a Membrane-targeted Nongenetic Photoswitch.

Mechanosensitive ion channels are present in the plasma membranes of all cells. They play a fundamental role in converting mechanical stimuli into biochemical signals and are involved in several physiological processes such as touch sensation, hearing, and blood pressure regulation. This protein family includes TWIK-related arachidonic acid-stimulated K+ channel (TRAAK), which is specifically implicated in the maintenance of the resting membrane potential and in the regulation of a variety of important neurobiological functions. Dysregulation of these channels has been linked to various diseases, including blindness, epilepsy, cardiac arrhythmia, and chronic pain. For these reasons, mechanosensitive channels are targets for the treatment of several diseases. Here, we propose a new approach to investigate TRAAK ion channel modulation that is based on nongenetic photostimulation. We employed an amphiphilic azobenzene, named Ziapin2. In the dark, Ziapin2 preferentially dwells in the plasma membrane, causing a thinning of the membrane. Upon light irradiation, an isomerization occurs, breaking the dimers and inducing membrane relaxation. To study the effect of Ziapin2 on the mechanosensitive channels, we expressed human TRAAK (hTRAAK) channels in HEK293T cells. We observed that Ziapin2 insertion in the membrane is able per se to recruit hTRAAK, permitting the exit of K+ ions outside the cells with a consequent hyperpolarization of the cell membrane. During light stimulation, membrane relaxation induces hTRAAK closure, generating a consistent and compensatory depolarization. These results add information to the Ziapin2 mechanism and suggest that membrane deformation can be a tool for the nonselective modulation of mechanosensitive channels.

A membrane intercalating metal-free conjugated organic photosensitizer for bacterial photodynamic inactivation.

Photodynamic inhibition (PDI) of bacteria represents a powerful strategy for dealing with multidrug-resistant pathogens and infections, as it exhibits minimal development of antibiotic resistance. The PDI action stems from the generation of a triplet state in the photosensitizer (PS), which subsequently transfers energy or electrons to molecular oxygen, resulting in the formation of reactive oxygen species (ROS). These ROS are then able to damage cells, eventually causing bacterial eradication. Enhancing the efficacy of PDI includes the introduction of heavy atoms to augment triplet generation in the PS, as well as membrane intercalation to circumvent the problem of the short lifetime of ROS. However, the former approach can pose safety and environmental concerns, while achieving stable membrane partitioning remains challenging due to the complex outer envelope of bacteria. Here, we introduce a novel PS, consisting of a metal-free donor-acceptor thiophene-based conjugate molecule (BV-1). It presents several advantageous features for achieving effective PDI, namely: (i) it exhibits strong light absorption due to the conjugated donor-acceptor moieties; (ii) it exhibits spontaneous and stable membrane partitioning thanks to its amphiphilicity, accompanied by a strong fluorescence turn-on; (iii) it undergoes metal-free intersystem crossing, which occurs preferentially when the molecule resides in the membrane. All these properties, which we rationalized via optical spectroscopies and calculations, enable the effective eradication of Escherichia coli, with an inhibition concentration that is below that of current state-of-the-art treatments. Our approach holds significant potential for the development of new PS for controlling bacterial infections, particularly those caused by Gram-negative bacteria.

Tamm Plasmon Resonance as Optical Fingerprint of Silver/Bacteria Interaction.

The incorporation of responsive elements into photonic crystals is an effective strategy for fabricating active optical components to be used as sensors, actuators, and modulators. In particular, the combination of simple multilayered dielectric mirrors with optically responsive plasmonic materials has proven to be successful. Recently, Tamm plasmon (TP) modes have emerged as powerful tools for these purposes. These modes arise at the interface between a distributed Bragg reflector (DBR) and a plasmonic layer and can be excited at a normal incidence angle. Although the TP field is located usually at the DBR/metal interface, recent studies have demonstrated that nanoscale corrugation of the metal layer permits access to the TP mode from outside, thus opening exciting perspectives for many real-life applications. In this study, we show that the TP resonance obtained by capping a DBR with a nanostructured layer of silver is responsive to Escherichia coli. Our data indicate that the modification of the TP mode originates from the well-known capability of silver to interact with bacteria, within a process in which the release of Ag+ ions leaves an excess of negative charge in the metal lattice. Finally, we exploited this effect to devise a case study in which we optically differentiated between the presence of proliferative and nonproliferative bacteria using the TP resonance as a read-out. These findings make these devices promising all-optical probes for bacterial metabolic activity, including their response to external stressors.

Insight on the Intracellular Supramolecular Assembly of DTTO: A Peculiar Example of Cell-Driven Polymorphism.

The assembly of supramolecular structures within living systems is an innovative approach for introducing artificial constructs and developing biomaterials capable of influencing and/or regulating the biological responses of living organisms. By integrating chemical, photophysical, morphological, and structural characterizations, it is shown that the cell-driven assembly of 2,6-diphenyl-3,5-dimethyl-dithieno[3,2-b:2',3'-d]thiophene-4,4-dioxide (DTTO) molecules into fibers results in the formation of a "biologically assisted" polymorphic form, hence the term bio-polymorph. Indeed, X-ray diffraction reveals that cell-grown DTTO fibers present a unique molecular packing leading to specific morphological, optical, and electrical properties. Monitoring the process of fiber formation in cells with time-resolved photoluminescence, it is established that cellular machinery is necessary for fiber production and a non-classical nucleation mechanism for their growth is postulated. These biomaterials may have disruptive applications in the stimulation and sense of living cells, but more crucially, the study of their genesis and properties broadens the understanding of life beyond the native components of cells.

Membrane Order Effect on the Photoresponse of an Organic Transducer.

Non-genetic photostimulation, which allows for control over cellular activity via the use of cell-targeting phototransducers, is widely used nowadays to study and modulate/restore biological functions. This approach relies on non-covalent interactions between the phototransducer and the cell membrane, thus implying that cell conditions and membrane status can dictate the effectiveness of the method. For instance, although immortalized cell lines are traditionally used in photostimulation experiments, it has been demonstrated that the number of passages they undergo is correlated to the worsening of cell conditions. In principle, this could impact cell responsivity against exogenous stressors, including photostimulation. However, these aspects have usually been neglected in previous experiments. In this work, we investigated whether cell passages could affect membrane properties (such as polarity and fluidity). We applied optical spectroscopy and electrophysiological measurements in two different biological models: (i) an epithelial immortalized cell line (HEK-293T cells) and (ii) liposomes. Different numbers of cell passages were compared to a different morphology in the liposome membrane. We demonstrated that cell membranes show a significant decrease in ordered domains upon increasing the passage number. Furthermore, we observed that cell responsivity against external stressors is markedly different between aged and non-aged cells. Firstly, we noted that the thermal-disordering effect that is usually observed in membranes is more evident in aged cells than in non-aged ones. We then set up a photostimulation experiment by using a membrane-targeted azobenzene as a phototransducer (Ziapin2). As an example of a functional consequence of such a condition, we showed that the rate of isomerization of an intramembrane molecular transducer is significantly impaired in aged cells. The reduction in the photoisomerization rate translates in cells with a sustained reduction of the Ziapin2-related hyperpolarization of the membrane potential and an overall increase in the molecule fluorescence. Overall, our results suggest that membrane stimulation strongly depends on membrane order, highlighting the importance of cell passage during the characterization of the stimulation tools. This study can shine light on the correlation between aging and the development of diseases driven by membrane degradation as well as on the different cell responsivities against external stressors, such as temperature and photostimulation.

Core-Shell Architecture in Poly(3-hexylthiophene) Nanoparticles: Tuning of the Photophysical Properties for Enhanced Neuronal Photostimulation.

This study shows that entirely thiophene-based core@shell nanoparticles, in which the shell is made of the oxidized form of the core polymer (P3HT@PTDOx NPs), result in a type II interface at the particle surface. This enables the development of advanced photon nanotransducers with unique chemical-physical and biofunctional properties due to the core@shell nanoarchitecture. We demonstrate that P3HT@PTDOx NPs present a different spatial localization of the excitation energy with respect to the nonoxidized NPs, showing a prevalence of surface states as a result of a different alignment of the HOMO/LUMO energy levels between the core and shell. This allows for the efficient photostimulation of retinal neurons. Indeed, thanks to the stronger and longer-lived charge separation, P3HT@PTDOx NPs, administered subretinally in degenerate retinas from the blind Royal College of Surgeons rats, are more effective in photostimulation of inner retinal neurons than the gold standard P3HT NPs.

Optical modulation of excitation-contraction coupling in human-induced pluripotent stem cell-derived cardiomyocytes.

Non-genetic photostimulation is a novel and rapidly growing multidisciplinary field that aims to induce light-sensitivity in living systems by exploiting exogeneous phototransducers. Here, we propose an intramembrane photoswitch, based on an azobenzene derivative (Ziapin2), for optical pacing of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The light-mediated stimulation process has been studied by applying several techniques to detect the effect on the cell properties. In particular, we recorded changes in membrane capacitance, in membrane potential (Vm), and modulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient Vm hyperpolarization followed by a delayed depolarization and action potential firing. The observed initial electrical modulation nicely correlates with changes in Ca2+ dynamics and contraction rate. This work represents the proof of principle that Ziapin2 can modulate electrical activity and contractility in hiPSC-CMs, opening up a future development in cardiac physiology.

Membrane Targeted Azobenzene Drives Optical Modulation of Bacterial Membrane Potential.

Recent studies have shown that bacterial membrane potential is dynamic and plays signaling roles. Yet, little is still known about the mechanisms of membrane potential dynamics regulation-owing to a scarcity of appropriate research tools. Optical modulation of bacterial membrane potential could fill this gap and provide a new approach for studying and controlling bacterial physiology and electrical signaling. Here, the authors show that a membrane-targeted azobenzene (Ziapin2) can be used to photo-modulate the membrane potential in cells of the Gram-positive bacterium Bacillus subtilis. It is found that upon exposure to blue-green light (λ = 470 nm), isomerization of Ziapin2 in the bacteria membrane induces hyperpolarization of the potential. To investigate the origin of this phenomenon, ion-channel-deletion strains and ion channel blockers are examined. The authors found that in presence of the chloride channel blocker idanyloxyacetic acid-94 (IAA-94) or in absence of KtrAB potassium transporter, the hyperpolarization response is attenuated. These results reveal that the Ziapin2 isomerization can induce ion channel opening in the bacterial membrane and suggest that Ziapin2 can be used for studying and controlling bacterial electrical signaling. This new optical tool could contribute to better understand various microbial phenomena, such as biofilm electric signaling and antimicrobial resistance.

Resonant Enhancement of Polymer-Cell Optostimulation by a Plasmonic Metasurface.

Organic semiconductors have shown great potential as efficient bioelectronic materials. Specifically, photovoltaic polymers such as the workhorse poly(thiophene) derivatives, when stimulated with visible light, can depolarize neurons and generate action potentials, an effect that has been also employed for rescuing vision in blind rats. In this context, however, the coupling of such materials with optically resonant structures to enhance those photodriven biological effects is still in its infancy. Here, we employ the optical coupling between a nanostructured metasurface and poly(3-hexylthiophene) (P3HT) to improve the bioelectronic effects occurring upon photostimulation at the abiotic-biotic interface. In particular, we designed a spectrally tuned aluminum metasurface that can resonate with P3HT, hence augmenting the effective field experienced by the polymer. In turn, this leads to an 8-fold increase in invoked inward current in cells. This enhanced activation strategy could be useful to increase the effectiveness of P3HT-based prosthetic implants for degenerative retinal disorders.

Azobenzene photoisomerization probes cell membrane viscosity.

The viscosity of cell membranes is a crucial parameter that affects the diffusion of small molecules both across and within the lipid membrane and that is related to several diseases. Therefore, the possibility to measure quantitatively membrane viscosity on the nanoscale is of great interest. Here, we report a complete investigation of the photophysics of an amphiphilic membrane-targeted azobenzene (ZIAPIN2) and we propose its use as a viscosity probe for cell membranes. We exploit ZIAPIN2 trans-cis photoisomerization to develop a molecular viscometer and to assess the viscosity of Escherichia coli bacteria membranes employing time-resolved fluorescence spectroscopy. Fluorescence lifetime measurements of ZIAPIN2 in E. coli bacteria suspensions correctly indicate that the membrane viscosity decreases as the temperature of the sample increases. Given the non-homogeneity and the anisotropy of cell membranes, as supported by the photophysical characterization of the probe within the lipid bilayer, we shed new light on the intricate membrane rheology.

Excited states engineering enables efficient near-infrared lasing in nanographenes.

The spectral overlap between stimulated emission (SE) and absorption from dark states (i.e. charges and triplets) especially in the near-infrared (NIR), represents one of the most effective gain loss channels in organic semiconductors. Recently, bottom-up synthesis of atomically precise graphene nanostructures, or nanographenes (NGs), has opened a new route for the development of environmentally and chemically stable materials with optical gain properties. However, also in this case, the interplay between gain and absorption losses has hindered the attainment of efficient lasing action in the NIR. Here, we demonstrate that the introduction of two fluoranthene imide groups to the NG core leads to a more red-shifted emission than the precursor NG molecule (685 vs. 615 nm) and also with a larger Stokes shift (45 nm vs. 2 nm, 1026 cm-1vs. 53 cm-1, respectively). Photophysical results indicate that, besides the minimisation of ground state absorption losses, such substitution permits to suppress the detrimental excited state absorption in the NIR, which likely arises from a dark state with charge-transfer character and triplets. This has enabled NIR lasing (720 nm) from all-solution processed distributed feedback devices with one order of magnitude lower thresholds than those of previously reported NIR-emitting NGs. This study represents an advance in the field of NGs and, in general, organic semiconductor photonics, towards the development of cheap and stable NIR lasers.