Symposium SB05—Light–Matter Interactions at the Interface with Living Cells, Tissues and Organisms

Developments in optogenetics and virology have demonstrated that modulating the neural activity of specific projections in the brain is capable of rescuing behavioral deficits, including those relevant to depression, autism and anxiety. This approach requires genetic modification of neurons via viral transduction of transducers of visible light, and thus light penetration depth is limited by the scattering of visible light (although fiberoptic interfaces can overcome this challenge). Additional channels for modulating neural activity would be valuable, ideally minimally-invasive. Here, we developed a non-genetic, nanoparticle-based approach to achieve projection-specific modulation of neural activity in freely behaving mice. First, we observed that gold nanorods were rapidly endocytosed into cultured neurons after just a few minutes of incubation. Transmission electron microscopy images illustrate that gold nanorods are located both inside and outside the endosome. Second, we showed that the endocytosed gold nanorods were transported retrogradely and anterogradely along axons, with average speed of 0.2 μm/s (1.7 cm/day). Third, we demonstrated the effectiveness of photothermal inhibition of neural activity with axon-transported gold nanorods, illustrating the concept of projection-specific modulation of neural activity in vitro, and also demonstrated biocompatibility of gold nanorods in neurons after one-week incubation and photothermal neural modulation. Finally, we observed the neural uptake and axonal transport of gold nanorods in vivo, as well as the effectiveness of neural modulation in mouse behavior. Overall, this nanoparticle-based methodology demonstrates a promising approach to non-genetic, projection-specific modulation of neural activity.

Bioluminescence (BL) has the potential to serve as a new “light” source to activate photosensitizers. Since excitation energy is delivered chemically, this approach can overcome the light-penetration problems of conventional photodynamic therapy (PDT) in deep tissue. We have developed novel RLuc8-Ce6 conjugates, which generate bioluminescence resonance energy transfer (BRET) from RLuc8, a Renilla luciferase mutant, to Chlorin e6 (Ce6), a photosensitizer, upon binding with methoxy-e-coelenterazine (meCTZ) substrates. A high BRET efficiency was achieved by optimizing the molecular ratio and spacing, as well as the spectral overlap. We also developed a protocol based on membrane fusion liposomes to deliver the protein constructs effectively into cells and obtained promising therapeutic effects of BL-PDT in a mouse model of metastatic, triple-negative breast cancer.

The technique of optocapacitance is a non-genetic way used to initiate action potentials in excitable tissues with light. The principle is simple: the current I_c flowing to the cell membrane capacitance C is given by I_c =CdV/dt + (V-V_s)dC/dt, where V is the membrane potential and V_s is the net surface potential. Membrane capacitance increases with an increase in temperature, therefore a fast increase in temperature will induce a (V-V_s)dC/dt term that, under conditions of current clamp, it depolarizes the membrane which open sodium channels thus initiating an action potential. The energy required to excite a neuron decreases as the pulse is made shorter. This is because the induced current depends on the rate of change of the temperature and not the temperature change. Therefore a long pulse does not contribute to the capacitive current thus becoming wasted energy. Experimentally, maximum efficiency is obtained with microsecond pulse durations (1). There are several ways to achieve this fast increase in temperature. An infrared pulse will increase the membrane temperature but the disadvantages are poor tissue penetration and increase of the tissue temperature (2). A more localized approach is to use light-to-heat energy transducers located in close proximity to the cell membrane. Gold nanoparticles, that have the advantage of surface plasmon resonance absorption, have been successfully used as such transducers to deliver just enough heat energy to quickly change membrane temperature of the neuronal membrane for the initiation of an action potential (3). In addition, gold nanoparticles can also be functionalized to get bound to a particular cell using antibodies (3) or specifically to the cell membrane using cholesterol (4). Other materials such as graphite particles, carbon nanotubes and mesoporous silicon (5) are also very good light-to-heat transducers that we have used to excite neurons. The technique has been applied to cultured dorsal root ganglion cells or to tissues such as brain slices (3) or isolated retina.
1. Carvalho-de-Souza JL, Pinto BI, Pepperberg DR, Bezanilla F.(2018) Optocapacitive Generation of Action Potentials by Microsecond Laser Pulses of Nanojoule Energy. Biophys J. 114(2):283-288.
2. Shapiro MG, Homma K, Villarreal S, Richter CP, Bezanilla F.(2012) Infrared light excites cells by changing their electrical capacitance. Nat Commun. 3:736.
3. Carvalho-de-Souza JL, Treger JS, Dang B, Kent SB, Pepperberg DR, Bezanilla, F.(2015) Photosensitivity of neurons enabled by cell-targeted gold nanoparticles. Neuron. 86(1):207-17.
4. Carvalho-de-Souza JL, Nag OK, Oh E, Huston AL, Vurgaftman I, Pepperberg DR, Bezanilla F, Delehanty JB.(2019) Cholesterol Functionalization of Gold Nanoparticles Enhances Photo-Activation of Neural Activity. ACS Chem Neurosci. 10(3):1478-87.
5. Jiang Y, Carvalho-de-Souza JL, Wong RC, Luo Z, Isheim D, Zuo X, Nicholls AW, Jung IW, Yue J, Liu DJ, Wang Y, De Andrade V, Xiao X, Navrazhnykh L, Weiss DE, Wu X, Seidman DN, Bezanilla F, Tian B. (2016). Heterogeneous silicon mesostructures for lipid-supported bioelectric interfaces. Nat Mater. 15(9):1023-30.

Light modulation of cell activity is an active field of research: the possibility of coupling low invasiveness and high resolution assisted the transition from a passive, diagnostic element to an active modulator of cellular physiology. The lack of natural absorbers led to the development of tools able to transduce the optical signal into a biologically readable one, typically through a combination of photo-thermal, photo-electrochemical and photo-capacitive effects. In this framework, organic materials, and in particular poly-thiophene based materials, demonstrated to be reliable for both in vitro and in vivo use [Feyen et al, 2016, Lodola et al, 2017, Zucchetti et al, 2016, Bossio et al, 2018].
In this work, we prove the behaviour of Poly-3-hexyl thiophene (P3HT) as active modulator of redox metabolic signalling. We first demonstrate that the photocatalytic activity of P3HT in aqueous environment is spatially and temporally confined to the illuminated area by means of Scanning ElectroChemical Microscopy (SECM). To study the possible interactions with the photoexcited polymer, in view of in vitro application, we focus our attention on two possible acceptor moieties present in the cell cytosol, namely cytochrome C and oxygen, being their energetic levels well aligned with those of P3HT. The former is a transmembrane protein located across the mitochondrial membrane, being one of the components of the cellular respiration cycle. Its biological relevance is thus associated with the metabolic functions of the cell. By coupling electrochemical and spectro-electrochemical methods, we demonstrate for the first time that a direct, photoinduced electron transfer does occur between P3HT and cytochrome C in extracellular, aqueous environment.
Our results shed light on a technique for active redox modulation of cell metabolism, through on demand, light-activated smart organic interfaces.

Optoelectronic wireless stimulation of cells and tissue is evolving as an inherently less invasive alternative to wired microelectrodes. Wireless stimulation is enabled by photoelectrodes that harvest the light excitation in a semiconducting layer and transduce it to a stimulating ionic current sufficiently strong to depolarize attached neurons thus allowing for optical triggering of action potentials. Organic semiconductors offer a range of important advantages for photoelectrodes such as biocompatibility and the possibility to process them on flexible plastic substrates.¹ In addition organic semiconductors can show an exceptional electrochemical stability in water and do not require a dielectric to separate the cell-containing electrolyte. As a result a high capacitive coupling is achieved between the semiconductor and the electrolyte. It is exploited in organic photocapacitors to stimulate efficiently retinal cells.² However, the absence of a dielectric can allow for direct charge transfer into the liquid causing faradaic currents and potentially cytotoxic reactive electrochemical species. Therefore the optimization of organic semiconductor based photoelectrodes requests the detailed understanding of the materials and device properties that impact on these different routes of ionic current generation.
Here we address the problem in organic photoelectrodes based on a planar p/n junction containing phthalocyanine (H2PC) and N,N′-dimethyl perylenetetracarboxylic diimide (PTCDI).² We combine the detailed characterization of photoelectrochemical currents with spectroscopic measurements and impedance spectroscopy. The data allows to establish a model that predicts quantitatively faradaic or capacitive current transients based on material properties and layer thicknesses. We find that most crucial to avoid faradaic processes are the semiconductor energy levels as well a precise tuning of electric fields present at the semiconductor liquid interface.

1. Hopkins, J. et al. Photoactive Organic Substrates for Cell Stimulation: Progress and Perspectives. Adv. Mater. Technol. 1800744, 1–10 (2019).
2. Rand, D. et al. Direct Electrical Neurostimulation with Organic Pigment Photocapacitors. Adv. Mater. 1707292, 1–11 (2018).

Electrical stimulation of tissue and ultimately individual cells has not only played an essential role in our understanding of the structure and function of excitable tissue but continues to serve as the basis for a variety of therapeutic interventions for the treatment of disorders ranging from cardiac arrhythmias to Parkinson’s disease. Advances in technology have attempted to overcome barriers associated with the spatial resolution (i.e., who and where to stimulate) and the invasiveness of the process. Optogenetics has revolutionized the way we can record and affect the electrophysiology of cells and tissue, using light as the input/output (I/O) interface. Though optogenetics has developed at a great pace and is making profound scientific contributions, the core of the technique requires genetic modifications of the cells or organism. This presents challenges both in terms of achieving targeted gene expression and the potential deleterious consequences of the expression of foreign proteins, which have implications on clinical translation to humans and regulatory approval. We report our developed breakthrough hybrid-nanomaterial synthesis process to enable minimally invasive, remote and non-genetic light-induced control of targeted cell activity with high spatial-temporal resolution. We combine one-dimensional (1D) nanowires (NWs) and two-dimensional (2D) graphene flakes grown out-of-plane with tailor-made physical properties for highly controlled photostimulation. Our non-genetic NW templated 3D fuzzy graphene (NT-3DFG) platform adds a powerful toolset to the basic scientists studying cell signaling within and between tissues, obviating the need for slow and expensive breeding protocols and/or the screening of viral serotypes to enable the use of light to control cell activity. As we continue to struggle to understand the cells and circuits involved in health and disease, our approach to controlling cell excitability has the potential to accelerate knowledge generation as well as the identification of novel therapeutic targets. Last, this platform can be adapted to address challenges in tissue engineering, i.e. the much-needed non-genetic stimulation control of engineered tissues. By controlled delivery of the NT-3DFG we will be able to locally and selectively control cellular activity with high spatial and temporal resolution of 3D tissues.

Organic nanoparticles provide a platform for two-photon microscopy and theranostics that has highly tunable spectroscopic properties and versatile biocompatibility pathways. Here we demonstrate two-photon absorption and subsequent photon upconversion in organic nanoparticles that utilize a three-body resonant energy transfer known as cooperative energy pooling (CEP). CEP is based on the sensitization of two-photon absorbing chromophores with low-energy antennae molecules in the surrounding environment. It relies on the simultaneous transfer of two singlet excitons, initially generated on the sensitizers, to a single high-energy exciton state on an acceptor. This provides for a vast increase in the effective two-photon absorption cross-section of the acceptor by placing real, as opposed to virtual, states at the low-energy resonance. It relaxes the requirement of coincident photons normally needed for two-photon absorption by temporarily storing the energy of one photon in a singlet exciton state. It also circumvents the need for intersystem-crossing events that occur in triplet-triplet annihilation (TTA) upconversion and therefore allows for the possibility of high energetic efficiency and short, nanosecond timescales for the upconversion process. Here we synthesize organic nanoparticles of sensitizer-acceptor mixtures with diameters on the order of 100 nm and with zeta potentials of approximately -50 mV. These carry out photon upconversion from red to blue light with energetic efficiencies of ~77% and on timescales of a few nanoseconds. We discuss the photophysical process, its efficiency, and its intensity dependence, and we suggest strategies for utilizing the concept in biological applications.

The photosynthetic enzyme reaction center (RC) is the photochemical core of the photosynthetic bacterium Rhodobacter sphaeroides, a bacterium able to grow using light as sole energy source. The RC is a transmembrane protein composed by three subunits and nine cofactors involved in a cascade of electron transfer reactions that, upon the absorption of a photon, produce a hole-electron couple. The electrical charges generated within the enzyme are roughly 3 nanometers apart from each other and have a lifetime that can last from tenths to few seconds. The generation of such nanocapacitor has a conversion photons to electrons efficiency close to unity, making this biological trabsduction appealing in principle for bioelectronics applications. [1] This biological macromolecule are also amenable for the assembly of organic-biological hybrids with improved enzymatic photoconvertion ability. [2-4]
Such photoconverters, either bare or as biohybrids, require an optimised interface with electrode surfaces for their applications [5]. Recently polydopamine (PDA), a self-assembling melanin-like bioinspired polymer, has been exploited to attach and protect enzyme into metal-organic frameworks (MOF) [4] and graphene nanosheets [5] on devices substantially maintaining their activities.
We present here PDA nanoparticles containing the photosynthetic RC able to confine the photoenzyme that retains unaltered the photoactivity, i.e. the capability to generate nanocondensers. The RC has been encased into PDA particles of different size from the micrometer to the nanometer, the smaller being characterised by a bright yellow luminescence. These PDA nanocontainers have an intrinsic n type semiconductive [6] making these organic RC containing nanocapsules highly interesting for bioelectronics purpose.

This work was funded by European Commission through the EU project 800926 - HyPhOE (Hybrid Electronics based on Photosynthetic Organisms)

[1] F. Milano, A. Punzi, R. Ragni, M. Trotta, and G. M. Farinola, Adv. Funct. Mater., 1805521, 1-17 (2018).
[2] A. Operamolla, R. Ragni, F. Milano, R.R. Tangorra, A. Antonucci, A. Agostiano, M. Trotta, G.M. Farinola, J. Mat. Chem. C, 25(3), 6471-6478 (2015)
[3] S. La Gatta, F. Milano, G. M. Farinola, A. Agostiano, M. Di Donato, A. Lapini, P. Foggi, M. Trotta & R. Ragni, Bioch. et Biophys. Acta (BBA)-Bioen., 1860 (4), 350-359 (2019).
[4] F. Milano, R. R. Tangorra, O. Hassan Omar, R. Ragni, A. Operamolla, A. Agostiano, G. M. Farinola & M. Trotta, Ang. Chemie Int. Ed., 51 (44), 11019-11023 (2012).
[5] M. Lo Presti, D. Vona, G. Leone, G. Rizzo, R. Ragni, S.R. Cicco, F. Milano, F. Palumbo, M. Trotta, G.M. Farinola, MRS Advances, 2019
[4] H. P. Peng, R. P. Liang, L. Zhang & J. D. Qiu, Biosen. Bioel., 42, 293-299 (2013).
[5] L. Zhou, Y. Jiang, L. Ma, Y. He, J. Gao, Appl. Biochem. Biotechnol. 175, 1007-17 (2015).
[6] P. F. Ambrico, A. Cardone, N. F. Della Vecchia, T. Ligonzo, S. R. Cicco, M. Mastropasqua Talamo, A. Napolitano, V. Augelli, G. M. Farinola & M. D'Ischia, J. Mater. Chem. C, 1 (5), 1018-1028 (2013).

Bismuth vanadate (BiVO₄) is a promising n-type semiconductor due to its superior photoelectrocatalytic performance, chemical stability, cost-effectiveness. Despite its beneficial advantages along with non-toxicity and biocompatibility, there are few studies for a biomedical application of BiVO₄. Here, we report a BiVO₄’s newly discovered capability to dissociate the β-amyloid (Aβ) aggregates associated with Alzheimer’s disease (AD) under illumination. An accumulation of β-sheet-rich amyloid aggregates in the brain is a major pathological hallmark of AD. The extremely high stability of amyloid structures makes it challenging to disassemble Aβ aggregates, which sparks the need for the development of new agents for breaking the pre-existing amyloid aggregates and alleviating Aβ-induced toxicity. The use of photoactive materials in medical application is an attractive strategy because of the temporal, spatial controllability, reduced side effects. The development of optogenetic technologies has opened up a new possibility of light-assisted treatment of AD by delivering light energy directly into the brain, which motivated us to develop a new photo-active anti-Aβ neurotoxin agent for localized therapy of AD. In this study, we have verified that nanoporous BiVO₄ photoelectrode can effectively break long Aβ aggregates into non-toxic, small-sized, and soluble Aβ species under the illumination of a white light-emitting diode and applied bias. According to our photochemical and microscopic analyses, photoexcited BiVO₄ photoelectrode under anodic bias generates oxidative stress, such as superoxide ions and hydrogen peroxide, which play a significant role of oxidizing Aβ peptides and irreversible disassembling the Aβ aggregates. The efficacy of photoelectrocatalytic disaggregation against Aβ assemblies was enhanced by doping Mo ions into BiVO₄ photoelectrode by improving electron-transport properties of BiVO₄. Furthermore, we have demonstrated that both pristine and Mo-doped BiVO₄ photoelectrodes are biocompatible and effective in reducing Aβ-induced cytotoxicity. Our work shows the potential of the visible light-active, nanoporous BiVO₄ photoelectrode-based platforms for dissociating the neurotoxic, highly stable self-assembled β-sheet-rich aggregates using light energy.

Diatoms are a large and prolific class of single cell photosynthetic microalgae, whose mesoporous biomineralized silica shells (frustules) encase the organic protoplasm [1].
Frustules exhibit interesting properties such as high surface area, mechanical resistance and mesoporosity, which make them appealing materials for applications in photonics, sensing, optoelectronics and biomedicine [2]. In particular, due to their quasi-periodic 3D hierarchical patterns of pores on both the nano- and microscale, frustules can act as natural photonic crystals.
We have demonstrated that in vivo incorporation of organic molecular emitters into frustules through diatoms’ silica biomineralization represents an efficient biotechnological route to new photonic materials whose properties result from the combination of the frustule hierarchical nanostructure with the luminescence of incorporated emitting molecules [3]. The in vivo incorporation approach has been also exploited by our group to generate porous biosilica nanostructures doped with organometallic iridium emitters [4].
More recently, we have also demonstrated that the in vivo incorporation of tailored light harvesting organic antennas into diatom cells can be a suitable method to enhance the algal photosynthesis, leading to increased diatoms growth rate and biomass production. In particular, our study points out that the combination of tailored photoactive molecules with diatoms microalgae can either represent a profitable strategy to get novel biohybrid photonic materials or be a straightforward non-genetic approach to enhance algal growth and biomass production.

[1] R. Ragni, S. R. Cicco, D. Vona, and G. M. Farinola, Green Material for Electronic (M. Irimia Vladu, E. D. Glowacki, N. S. Sariciftci and S. Bauer, Wiley VCH Verlag GmbH & Co. KGaA), 287-313 (2017).
[2] R. Ragni, S. R. Cicco, D. Vona and G. M. Farinola, Adv. Mater., 1704289, 1-23 (2017).
[3]R. Ragni, F.Scotognella, D.Vona, L.Moretti, E.Altamura, G.Ceccone, D.Mehn, S.-R.Cicco, F.Palumbo, G.LanzaniandG. M.Farinola,Adv. Funct. Mater., 28(24), 1706214-1706223(2018).
[4] G. Della Rosa, D. Vona, A. Aloisi, R. Ragni, R. Di Corato, M. Lo Presti, S. R. Cicco, E. Altamura, A. Taurino, M. Catalano, G.-M. Farinola and R. Rinaldi, ACS Sust. Chem. & Eng.,7(2), 2207-2215 (2018).

In this talk, I will discuss the chemical approach to prepare functional surface of conjugated polymers for interaction with biological system. Polymers exhibiting photovoltaic effect will be discussed in relationship with the potential of light-induced interaction with biological systems.

Composites, in which two or more material elements are brought together to provide properties unattainable by single component alternatives, have a long historical record dating back to ancient times. Few of them have included a living microbial community as a key design element. Here, we describe the use of a self-doped conjugated polyelectrolyte, namely CPE-K, in combination with Shewanella oneidensis to generate gels that can produce biologically-derived currents. CPE-K is a key ingredient due to its ability to dope via protonation reactions and the stability of the resulting polarons under biologically relevant conditions, i.e. buffered aqueous media. Moreover, CPE-K forms gels that exhibit both ionic and electrical conductivity and permit the diffusion of nutrients/waste to/from microorganisms entrapped within the gel network. One finds that the biocomposite gels can generate more than two orders of magnitude greater biocurrent density, when compared to a standard Shewanella oneidensis biofilm formed atop gold electrodes. Such an increase reflects the increase in dimensionality of the biotic/abiotic interface from two to three. We will also provide characterization of the device characteristics and will discuss the vitality of the microbial community within the gels.

We present here the development of a physical human eye model – based on hybrid soft/rigid materials - in order to create a test bench reproducing the optical eye properties.
We have developed phantom eye tissues in order to mimic the different parts like the sclera or the ciliary body as finely as possible regarding the optical properties. As a matter of fact, these parts are crucial in order to mimic human eye but often neglected in the literature. For the development of these models, we used the optical parameters (absorption and scattering coefficients; and refractive index) extracted from an experimental study carried out on porcine eyes – which are close to human one. Moreover, we present a soft actuated model of the iris where the aperture ranges from 1 mm to 8 mm. Finally, all the different parts are put together to obtain a device mimicking exactly the optical properties of an eye. Our work encompasses eye optical simulation, soft material for fabrication and characterization of the eye phantom model.
Light stimuli are used in the medical field to treat diseases as glaucoma based on infrared light or used blue light to treat circadian sleep disorders. The models developed in this study allow us a better understanding of the amount of light that propagates inside the eye.

Organic electronic ion pumps (OEIP) are an emerging bioelectronic medicine technology for on-demand delivery of pharmacologically-active species. While electrical control is advantageous for providing precise spatial, temporal, and quantitative delivery, it necessitates wiring, which complicates implantation. Herein we demonstrate integration on a flexible carrier of an OEIP with a photovoltaic driver which can be addressed by red light within the tissue transparency window. Organic thin-film bilayer photovoltaic pixels are arranged in series and tandem to provide the 2-5 V necessary for operating the high-resistance electrophoretic ion pumps. We demonstrate light-stimulated transport of protons as well as acetylcholine. The end result of our work is a thin and flexible integrated wireless device platform for targeted drug delivery.

Stimulating and recording the electrical activity of neurons and muscles is a major scientific and technological challenge. Although this field dates back centuries, it still remains at the forefront of contemporary investigations, in particular owing to the exploding interest in brain machine interfaces and electronics skin technology. Our investigations in recent years focus on developing and characterizing highly efficient nanomaterial based platforms for superior electronic interfacing with the human body. In particular, we focus on artificial vision and skin electronics for recording emotions. Artificial vision in particular is a very active field with many researchers and companies are trying to restore vision to blind patients through specially designed electronic devices. We recently implemented and validated ex-vivo, two novel systems: The first consists of photosensitive pixels made of a three-dimensional matrix of carbon nanotubes (CNTs) decorated very densely with quantum dots (QDs) or nano rods (NRs) (referred to also as quantum rods (QRs)). Light is absorbed and converted into a transient electrical dipole by the QRs/CNTs system. The use of a three-dimensional matrix as well as an optimized selection of QRs, their surface coating, and conjugation procedure contributes to the superior properties of our films. The second system consists of organic pigments. These materials offer superior photo response and most importantly can be readily implemented on flexible materials. Compared with other photo-sensitive artificial retinal platforms the two systems we have developed so far are marked by several clear advantages. Foremost is low stimulation threshold. Second is their biocompatibility, chemical stability and mechanical flexibility rendering the devices improved stability in the tissue. Implementing some insights gained in our artificial retina project into the realm of skin electronics, we developed a new skin electromyography system to open entirely new and exciting opportunities in recording facial emotions, in neuro-modulation application and in bio-feedback based on EMG.

Organic electronics has recently emerged as a powerful technology platform for bio-sensing applications. In many applications, the organic devices are operated in direct contact with an aqueous environment, thus resulting in strong coupling between charge transport in the organic solid and water dipoles, ions and cells in the liquid. These interactions, although dictate the sensing performance, are not well understood. In particular, how these interactions are influenced by the structural and morphological properties of the organic materials remains unknown. In terms of sensing mechanisms, many devices make use of only the electrical responses of organic semiconductors, while the optical signatures of these materials, which are very sensitive to their surroundings, have not been utilized.
In this talk, I will introduce how charge accumulation spectroscopy (CAS) can be applied with other characterization techniques to study the structure-charge transport property relations at the water-solid interfaces. In the first example, we exploit water-gated organic field-effect transistor as the testing platform to investigate the structure-dependent localization of polaronic charge carriers at the organic semiconductor-liquid interface. Our results reveal that the degree of charge delocalization is reduced drastically when the charge carriers are moved from the bulk semiconductor to the semiconductor-water interface, suggesting the existence of a highly disordered surface layer in contact with water. It is also found that the charge delocalization could be further reduced by intercalation of chloride ions (from saline solution) in the semiconductor surface layer. This study suggests that the spectroscopic signatures of polaronic charge carriers could be a sensitive probe to detect the structure-dependent charge localization at organic solid-liquid interfaces. In the second example we combine electrochemical quartz crystal microbalance (EQCM) with CAS to study the ion-to-electron conversion efficiency and water uptake properties of the active layer of organic electrochemical transistors (OECTs). Two material systems, namely PEDOT:PSS (representing depletion-mode OECT) and p(g2T-TT)(representing accumulation-mode OECT), are investigated. We found that the ion-to-electron conversion efficiency of both p(g2T-TT) and cross-linked PEDOT:PSS is close to one at low voltage, indicating highly efficient doping and de-doping processes; while the coupling tends to be weaker at higher voltages, suggesting reduction in the doping efficacy at high ion concentrations. The study also reveal that the PEDOT:PSS layer with low or no cross-linkers exhibits low ion-to-electron conversion efficiency, which is likely associated with the high density of trap states for cations. By simultaneously measuring the mechanical, electrical and optical properties of the polymeric active layer, we are also able to estimate the water hydration numbers of the injected ions. The result suggests that due to the hydroscopic nature of the PSS phase, the PEDOT:PSS film becomes increasingly viscous and hydrated upon ion injection at high operation voltages.

Photo-crosslinking hydrogels have been vastly utilized as scaffolds for tissue engineering and regenerative medicine. Albeit, ultraviolet and visible light, which has been shown strongly intervene with biological systems, are currently utilized as major excitation sources for hydrogel photo-crosslinking methods. Moreover, these excitation wavelengths are highly limited for in vivo applications because of the compromised spatial resolution and penetration depth. Alternatively, near-infrared (NIR) lights with minimal cellular and tissue interactions become a better candidate. Given the significance of NIR light in next-generation photo-crosslinking hydrogel for regenerative medicine and tissue engineering, a NIR-mediated photo-crosslinking and post-gelation modification method is on demand. Recently, with the swift development of upcovnersion nanoparticle (UCNP), emerging efforts have been made to drive various photoreactions with NIR light, for instance photo-cleavage, photo-isomerizations, photo-click reaction, and photo-polymerization. In terms of hydrogel crosslinking, the incorporation of NIR light as external stimulus is limited to photothermal crosslinking. The photothermally induced crosslinking mechanism requires high intensity NIR light for generating chemical reactions, where significant heating effect is inevitable. In theory, a NIR photo-crosslinking mechanism without utilizing heat as energy source will be more desirable for biomedical applications. Interestingly, photochemically activated initiator is rarely explored for NIR based hydrogel crosslinking systems.
This study demonstrates NIR-mediated photo-crosslinking (NmPC) of functionalized 4-arm-PEG with small molecule cross-linker, in which the interaction between NIR (980nm) excitation and UCNPs activate a photo initiator, resulting in stimulating photo-click reaction to form hydrogel. In order to utilize NIR excitation more efficiently, multi-shelled UCNPs were synthesized according to a reported procedure. As the efficient NIR (980nm) to visible (520nm-540nm) converter, the multi-shelled size increase was confirmed with TEM (30-60nm) and a 19 times luminescence enhancement was confirmed with luminescence spectroscopy. From rheometry characterization, the NIR mediated hydrogel cross-over point (G’=G’’) was found to be 43 seconds under 15 W/cm² 980nm laser intensity and Young’s moduli was found to be 1.9 kPa. Typical porous hydrogel structures were confirmed with SEM and HIM. Adipose-derived Mesenchymal Stem Cells (ADMSCs) were seeded on NmPC hydrogel with different surface binding ligand (RGD) density, showing great cellular viability and different spreading behaviors. Based on the hydrogel mechanical property, different chondrogenic behaviors were observed depending on different surface RGD functionalization, where low density (2mM) shows better chondrogenic markers expression comparing to high density (10mM) and conventional tissue culture polystyrene (TCPS). Interestingly, a proof-of-concept demonstration of NIR-mediated hydrogel printing was succeeded using same hydrogel precursor composition and focused 980nm laser beam.
By utilizing the upconversion effect of UCNPs to convert NIR light into visible emission, 4-arm-PEG chains could be crosslinked through photo-click reaction mediated by the photo initiator. To generate a 2D culture environment for ADMSCs differentiation, various concentrations of RGD ligands were functionalized on the hydrogel surface using NIR light. Low density RGD surface functionalization showed better chondrogenic differentiation comparing to high density and TCPS. With further improvement on the efficiency of upconversion process and better photo-initiator for the photo-click reaction, this hydrogel system can be a powerful approach for in vivo hydrogel property manipulation, cell/protein delivery, and tissue engineering applications.

In living systems, color is generated in three main ways - through bioluminescence, as seen in dinoflagellates; through pigments that absorb and reflect light, such as in human skin; or through specific structures that combine light interference and diffraction, such as in peacock feathers. The latter two phenomena do not involve light emission, but rather interact with light to yield an impressive range of colors. Many structural colors also involve the use of pigments, including melanins, that are patterned to geometrically interact with light and thereby enhance the phenomenon. Melanins are a group of organic pigments widely found across the kingdoms of life and are best known for their protective qualities against ultraviolet radiation. Eumelanin, responsible for light brown to black coloration, forms melanosomes in several types of bird feathers, which consequently organize to form structural color with and without iridescence, while simultaneously protecting from UV light, dissipating heat, and minimizing misdirected scattering.
This useful combination of protective and tunable characteristics has long been pursued across the fields of biochemistry and materials science. There has been great interest in controlled synthesis of color using pigment-related structural color, as the applications of such biocompatible and visually dramatic materials in products such as passive displays, inks, or cosmetics, become more evident. Only a handful of methods have thus far been published, the most prominent of which involve the use of high refractive index melanin or melanin-like materials as the shell layers or core of silica or polystyrene particles. Other methods include incorporation of polydopamine or synthetic melanin into photonic crystals, or thin films. In all of these approaches, the focus has largely been on incorporating eumelanin or a similar synthetic analog such as polydopamine. However, there are other types of pigments, such as pheomelanin or carotenoids, that may also be involved in structural color in Nature. In contrast to eumelanin, these pigments absorb at different spectra and thus may expand the toolbox of synthetic, protective, and biocompatible structural colors. Furthermore, combinations of pigments within multi-layer structures that utilize materials with both high and low refractive indices can create additional complexity with regard to light interaction, pointing towards optical assemblies that yield greater precision, efficiency, reflectance, and finer tunability.
Here we explore the incorporation of mixtures of synthetic eumelanin, pheomelanin, and carotenoids in multiple layers of synthesized particles to yield combination pigment-structural colors. We will examine criteria such as multi-layer shell coating, stacking of close-packed layers of pigment-incorporated particles, shell thickness, and shell composition, and heterogeneity of structures. Properties such as reflectance, absorption, packing, layer interfaces, and global architectures will be studied using a variety of methods, including spectrophotometry, scanning and transmission electron microscopy, light microscopy, and angular spectroscopy. In doing so, we hope to provide a path towards the utilization of the impressive protective properties and functions of natural pigments and their synthetic analogs in the study of structural color, resulting in multi-functional, biocompatible, and scalable coloration.

Graphene is a two-dimensional (2D) material consisting of a single sheet of sp2 hybridized carbon atoms laced in a hexagonal lattice, with potentially wide usage as a Raman enhancement substrate, also termed graphene-enhanced Raman scattering (GERS), making it ideal for sensing applications. GERS improves upon traditional surface-enhanced Raman scattering (SERS), combining its high sensitivity and spectral fingerprinting of molecules, and the unique advantages of graphene including simple processing, superior uniformity and low cost. This enables fast and highly sensitive detection of a wide variety of analytes. Accordingly, GERS has been investigated for a wide variety of molecule sensing applications. In the field of molecule sensing, biomolecule detection is an important branch and plays significant roles in biomedical research and medical diagnosis. While GERS have witnessed great success in the sensing of small organic molecules, its potential in biomolecule sensing is yet to be discovered. In this work, we report our pioneer investigations in using GERS for biomolecule detection, including various types of proteins. The unique molecular selection rules in GERS of small molecules still apply for proteins. In addition, we revealed several structures in proteins that enable strong GERS signals. Furthermore, the application of GERS for tissue imaging show unprecedentedly high signal-to-noise ratio, high spatial resolution, rapid measurement and multiplexed data obtained. We further demonstrate the effectiveness of GERS-based hyperspectral imaging as a novel tool for disease diagnosis. Our work unveiled the tremendous potential of GERS in biomedical studies and opened a new path of biosensing using GERS,

Efficient Plasmon enhanced photonic pan-microbial pathogen inactivation was demonstrated under conditions that allow for shockwave generation and E-field mediated cavitation. We report ultra-fast transient absorption of gold nanoparticles to explore the microscopic mechanism of shockwave generation. We use 800nm, 100fs pulses with a repetition rate of 1KHz generated with chirped pulse amplification as the pump beam and a white light continuum derived from the pump beam incident on a sapphire plate as the probe beam. Transient absorption spectra were obtained by chopping the pump beam at 500Hz with lock-in detection. Systematic studies of probe wavelength, intensity, and nanoparticle morphology to test the proposed mechanism of inactivation. Our work demonstrates the potential of plasmonic nanomaterials for pan-pathogen inactivation without targeting.

Near infra-red (NIR) light mediated photothermal therapy (PTT) has emerged as an excellent therapeutic method to ablate cancer due to its outstanding benefits of noninvasiveness, high specificity and high tissue transparency. Along with therapeutic treatment, diagnosis of tumors is equally vital for the visualization of the tumor and approaching therapeutic activity of remotely activated nanoparticles. Among several imaging modalities, NIR fluorescence imaging is an excellent technique due to its high sensitivity, low scattering, portability and safety. Recently, conjugated polymer nanoparticles have successfully employed as biological imaging agents and more recently evolved for photothermal therapy.
Our lab has recently demonstrated that the donor acceptor based conjugated polymer poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe) is an excellent photothermal therapy agent but the oligomer of PCPDTBSe as fluorescent material has not yet been explored. Oligo PCPDTBSe showed emission in the NIR region (800 nm) upon excitation at 550 nm. The NIR fluorescence of the oligomer was explored for cellular imaging along with photothermal therapy when combined with the high molecular weight (hMW) segment of PCPDTBSe in 2:1 (oligo: hMW) ratio to form hybrid donor acceptor polymer particles (HDAPPs). Oligo NPs and hMW NPs were also prepared from oligomer and hMW of PCPDTBSe respectively by similar method for comparison. HDAPPs showed absorption maxima at 550 nm and 760 nm in UV-visible spectroscopy due to the presence of oligomer and hMW PCPDTBSe respectively, along with a new peak at 650 nm because of combined absorption. HDAPPs displayed a huge stokes shift of 264 nm with emission maxima at 810 nm upon excitation at 550 nm, producing 0.077 quantum yield. The hydrodynamic diameter of HDAPPs was found 65 nm, whereas oligo NPs and hMW showed 107 nm and 94 nm respectively. Upon 808 nm laser (3W) irradiation, 100 µg/ml hMW NPs produced temperature increase to 45°C, whereas equal concentration of HDAPPs produced temperature increment to 40°C; which is an unexpected phenomenon as HDAPPs contain only 1/3 of the heat generating hMW PCPDTBSe. HDAPPs were photostable with minimal decrease in fluorescence intensity through consecutive heating cycles. No significant decrease in heat generation capacity or fluorescence ability of HDAPPs was found despite of autoclaving for sterilization. HDAPPs incubated with non-tumorigenic MCF10A breast cells and triple negative MDA-MB-231 breast cancer cells displayed no cytotoxicity up to 100 µg/ml in the absence of NIR exposure. HDAPPs were clearly visible in the cytoplasm around the perinuclear region of MDA-MB-231 cell lines in Fluorescence microscopy. Upon 808 nm laser (3W) irradiation, MDA-MB-231 cells incubated with 40 µg/ml HDAPPs produced 35% cell viability whereas 100% cell killing was observed with 100 µg/ml HDAPPs. These results demonstrate that HDAPPs are a novel theranostic photothermal agent composed of two different molecular weights of PCPDTBSe for imaging as well as ablation of cancer cells.

We further our understanding of the optical properties of squid chromatophore pigment in order to interrogate its utility as a functional optical coating. Cephalopods (which include octopuses, cuttlefish, and squid) have evolved to create one of the most complex photonic systems. Using hierarchical layers of optical organs, these animals can change the color patterns of their skin within milliseconds. A majority of their bulk coloration is facilitated by pigmentary organs known as chromatophores, which can expand many times their size in surface area. The pigment responsible for this color has been identified as a mixture of xanthommatin and decarboxylated xanthommatin in previous studies, and its refractive index has been measured in both the solution phase and aerosol phase.
The indices of refraction of the pigments are needed for essential analysis and modeling of the chromatophore organs in order to understand how the cephalopod accomplishes its unique color and pattern changes for camouflage. To examine these optical properties in the solid state, we fabricated thin films using extracted squid chromatophore pigment mixed with polyvinyl alcohol (PVA) in a 50/50 mixture by weight. The pigment/polymer solution was spin-coated onto silicon substrates achieving a range of thicknesses (200 – 700 nm) by controlling the spin speed. The polymer aids in the homogenous distribution of pigment throughout the film by eliminating aggregation and crystallization. This method has resulted in improved ellipsometry measurements, which require very smooth films, and thus can model the pigment’s refractive index from visible wavelengths up to 3 microns. Using the procedures in this study, we demonstrate increased control of thin-film fabrication methods of extracted pigments, and a greater understanding of their optical utility within the animal, for photonic devices, or coatings.

Optogenetics and covalent approaches to bio conjugation allow to achieve large and effective cell photo-stimulation, yet these are invasive methods that might encounter severe limitations on the way towards clinical applications in photopharmacology. The non-covalent affinity of photoresponsive molecules to biotargets represents an attractive alternative. Here, we show that an amphiphilic azobenzene photochromic molecule, fully locked in water, recovers its photo switching dynamics once screened by the plasma phospholipid system. According to our steady state and time resolved spectroscopic investigations the photophysical scenario in different media is dramatically different. In molecular aggregates formed in water the isomerization reaction is hindered while the radiative deactivation is enhanced by an excimer type transition. Once protected by a lipid bilayer, either in artificial micelles or in the cell membrane, the photochromic molecules reacquire their photoisomerisation capacity. Together with the natural affinity for the plasma membrane, this suggests a potential in cell opto-stimulation. We demonstrate in vitro the reversible modulation of the membrane potential via illumination with visible light. These data represent a new rationale for designing photoresponsive systems that operate via simple non-covalent affinity to biotargets and can be of importance for future applications in photopharmacology.

Realizing soft, conformable, free-form opto-bioelectronic devices is of great interest in the wearable and bio-compatible medical device industries [1-2]. To achieve superior wearable and bio-compatiable photomedical devices, high-performance and highly reliable free-form optoelectronic devices should be manufactured to accommodate a wide range of soft materials and shapes. In addition, a conformable surface light source should be directly attached to the skin to achieve a high-performance therapeutic effect. However, most studies have reported limited opto-bioelectronic devices that can only be fabricated on specific materials and shapes, or have relatively low performance and reliability. Even wearable photomedical device studies are mostly non-contact approaches based on point light sources, which are not flexible. Few studies on skin-attachable phototherapeutics based on ultimately soft, conformable, free-form optoelectronic devices have been reported.
In this study, we report soft, conformable, free-form OLEDs that can be attached to the skin for effective phototherapeutics. Ultra-thin free-form OLEDs (10 μm) are sandwiched by an attachable barrier and can be transferred to any soft, conformable material, including the skin. The attachable barrier is made of a nano-laminate film composed of three dyads of ZnO, Al₂O_3, and SiO₂ polymer to have high barrier performance (1.2 x 10^-5 g/m²/day) and reliability to prevent a phase transition to boehmite even in moisture such as sweat [3]. Therefore, free-form OLEDs transferred to any soft material such as the skin and textiles showed high performance (>20mW/cm², 80 cd/A), low voltage (<10V), folding reliability, long operation reliability (>100h), and washing reliability. When these free-form OLEDs were applied to real human skin keratinocytes and fibroblasts, cell proliferation was stimulated (>25%) and cell migration was effectively enhanced (>30%). Also, when the free-form OLED was attached to the rats' skin wound, the skin area was increased (>10%) and re-epithialization was improved (>20%). In conclusion, it is expected to be applicable to various skin-attachable phototherapeutics based on high-performance, high-reliability, bio-compatible free-form OLEDs.

[1] Y. Jeon, H. Choi, M. Lim, S. Choi, H. Kim, J. H. Kwon, K. Park K.C. Choi, Advanced Materials Technologies, 2018
[2] Y. Jeon, H. Choi J. H. Kwon, , S. Choi, K. Park K.C. Choi, Society for Information Display 2018 International Symposium, 2018
[3] E.G. Jeong, Y. Jeon, S.H. Cho, K.C. Choi, Energy & Environmental Science, 2019

The modern healthcare and biomedical systems show a clear trend towards personalized, predictive, and preventive medicine. Development of the concept, commonly known as mobile health (mHealth), means that a huge shift in the paradigms of medical device architectures is to be expected in the near future thanks to the increased portability of medical devices as well as increase in number of specific mobile-based apps. An ideal wearable device should possess a set of important requirements, such as (i) low cost of fabrication, (ii) being conformable and compatible with human skin, and (iii) multifunctionality. The latter is of special importance if the goal is to build not just a single specific device, but to rather develop a technology and basis for scalable fabrication of devices that are capable to detect a plurality of vital signals (HR, EEG, ECG, hydration, galvanic response, etc.).
In order to develop the universal technology that meets all three requirements mentioned above, we propose to utilize graphene in combination with epidermal technology. The conventional epidermal biosensors are based on metal and silicon based thin films that are patterned into special structures for softness and stretchability and embedded into soft biocompatible polymers. The choice of two-dimensional materials is the most natural due to their ultra-thinness, allowing extreme flexibility, transparency, and conformability to almost any rough surface, including skin [1]. Graphene based passive electrodes have been successfully used to epidermal sensing of electrocardiograms (ECG), electromyogram (EOG), electroencephalogram (EEG), skin temperature, and skin hydration [1], [2]. It is important to emphasize that the research work is based on large-area CVD-grown graphene, allowing us to develop low-cost, wearable, and fully conformable to skin devices. Furthermore, large area fabrication gives an ultimate promise for future devices fully based on 2D materials to be available on market. In terms of possible applications, the proposed technology can be easily expanded towards other fields of healthcare biosensing, such as in vivo electrophysiology, UV exposure sensing, pressure sensing, or even towards building electronic skin, and prosthetics.

[1] S. Kabiri Ameri et al., “Graphene Electronic Tattoo Sensors,” ACS Nano, vol. 11, no. 8, pp. 7634–7641, Aug. 2017.
[2] S. K. Ameri et al., “Imperceptible electrooculography graphene sensor system for human–robot interface,” npj 2D Mater. Appl., vol. 2, no. 1, pp. 1–7, 2018.

One of the major challenges in deciphering the fundamental principles of cognition is the lack of appropriate tools for seamless interfacing with neurons across all their signaling modalities. Gaining holistic understanding of neural circuits and their control of behavior requires invention of neural probes that can simultaneously record and modulate electro-chemical activity of neurons while evoking minimal inflammatory response for periods ranging from minutes to years. Multifunctional fibers have recently emerged as a promising platform for integrating multiple functional elements to probe and control neural activity that also minimizes the foreign body response.

In my presentation, I will describe our efforts in further expanding the multifunctionality of polymer-based fiber probes by incorporating an electrochemical sensor that enables real-time tracking of neurotransmitter dynamics in behaving animals. This is achieved by introducing a carbon nanotube (CNT) based electrocatalytic electrode within the multifunctional fiber-based probes during their fabrication via thermal drawing process. The resulting devices can be implanted chronically and perform electrical recording and stimulation of neurons, light delivery through waveguides for optogenetics, drug and gene delivery via microfluidic channels, and voltammetry via the CNT electrodes for dynamic detection of dopamine. We envision that these multimodal, miniature, and mechanically compliant probes will facilitate understanding of the neurophysiological underpinnings of dopamine-dependent behaviors including reward, addiction, and motor control.

Nanoscale materials enable unique opportunities at the interface between the physical and life sciences, for example, by integrating nanoelectronic devices with cells and/or tissue to make possible bidirectional communication at the length scales relevant to biological function. In this presentation, I will overview a new paradigm for seamlessly merging electronic arrays with the brain and other key components of the nervous system in three-dimensions. First, I will discuss the design considerations of matching structural, mechanical and topological characteristics of neural probes and brain tissue, thus leading to mesh electronics systems that are immune-privileged and enable uniquely stable electrophysiology such that it is possible to track and stably record from the same single neurons and neural circuits on the time scale of at least year. Second, I will describe a selection of new opportunities using the mesh electronics paradigm, including (i) nonlinear lesion-free implantation in the retina and brain, (ii) development of new nanoelectronic devices for subcellular resolution recording, and (iii) and advances in interfacing that can enable scalable recording and stimulation of large numbers of neurons. I will conclude with discussion of opportunities and challenges pushing towards tools that can significantly advance fundamental neuroscience and electronic medicine in humans.
Selected References
(1) http://meshelectronics.org/
(2) T.-M. Fu, et al., Nat. Methods 13, 875-882 (2016).
(3) G. Hong, et al., Science 360, 1447-1451 (2018).
(4) X. Yang, et al., Nat. Mater. 18, 510–517 (2019).
(5) G. Hong & C.M. Lieber, Nat. Rev. Neurosci. 20, 330–345 (2019).

Mammalian brains consist of billions of neurons operating at millisecond time scales, which current recording techniques only capture a tiny fraction. Recent advances in CMOS device design have led to high-recording quality planar probes, with diminishing sizes to ameliorate the extent of tissue damage. Matching these powerful silicon electronics to the inherently three dimensional architecture of the brain has remained challenging however, as devices are constrained to the planar two dimensional surfaces required for silicon processing. Here we describe a chronic interface using arrays of microwires read out by CMOS-based devices with a low-tissue damage, and controllable, three dimensional distribution of recording sites. The core concept is using a bundle of insulated microwires mated to a large-scale CMOS microelectrode array, such as found in modern camera chips or displays. We show recent results on the mechanics and tissue damage from microwire insertion scales strongly with wire diameter. Microwires with <25µm diameters are shown to have minimal to no vascular disruption or bleeding, as opposed to more conventional 75 to 100 µm devices. These microwires are then arranged into bundles to control the spatial arrangement and three dimensional structure of the distal (neuronal) end, while providing a robust parallel contact plane on the proximal side which is interfaced to a planar pixel array. The modular nature of the design enables a wide array of microwire types and size to be mated to a variety of different CMOS chips, making the same fundamental platform scalable from a few hundred electrodes to tens of thousands. We thus link the rapid progress and power of commercial multiplexing, digitisation and data acquisition hardware together with a bio-compatible, flexible and sensitive neural interface array. We present recent massively parallel recording using mouse and rat models, showing both spiking activity from single neurons and local field potentials within both chronic and acute settings.

The interaction between the cell membrane and the measuring electrode is crucial for crucial for sensitive measurement of cell electric activities. We are interested in exploring nanotechnology and novel materials to improve the membrane-electrode coupling efficiency. Recently, we and other groups show that vertical nanopillars protruding from a flat surface support cell survival and can be used as subcellular sensors to probe biological processes in live cells. The nanopillar electrodes deform plasma membrane inwards and induce membrane curvature when the cell engulfs them, leading to a reduction of the membrane-electrode gap distance and a higher sealing resistance. As an electrode sensor, nanoelectrodes offer several advantages such as high sensitivity, subcellular spatial resolution, and precise control of the sensor geometry. Furthermore, we found that the high membrane curvature induced by nanoscale electrodes significantly affects the distribution of curvature-sensitive proteins and stimulates several cellular processes in live cells. Our studies show a strong interplay between biological cells and nanoscale topography, which is an essential consideration for future development of interfacing devices.

In this talk, I will discuss several projects related to engineering conductive materials and developing fabrication methods to allow electronics with effective electrical interfaces with biological systems, through tuning their electrical as well as mechanical properties. The end result is a soft electrical interface that has both low interfacial impedance as well as match mechanical properties with biological tissue. Several applications of such electronics will be presented.

Bioelectronic medicines targeted at the peripheral nervous system have the potential to address a wide variety of diseases, from diabetes to bladder dysfunction. The foundational concepts have existed for decades but implementation has been limited and fraught with persistent challenges, including lack in target specificity, nerve interface biofouling, and inability to acquire real time physiologic signals for conditional delivery of corrective stimuli, leading to excess and unnecessary stimulation. Here we present a set of materials, a treatment strategy and supporting technology platform that address many of these challenges, using bladder control as model system. Specifically, we report capabilities for continuous monitoring of bladder function using an ultralow modulus, stretchable strain gauge to measure dimensional changes, real-time data analytics to identify pathological behavior based on the resulting data, and automated, closed-loop optogenetic neuromodulation of bladder sensory afferents to normalize bladder function in the context of acute cystitis, with generic applicability to many other organ systems and conditions.

Miniature, wireless bioelectronic devices enable less invasive surgical implantation and the ability to target tiny nerves or brain areas. However, as these neural stimulators become smaller, we must engineer new ways to deliver power. Conventional power deliver relies on long wires to deliver power from an implanted battery or subcutaneous antenna. These leads can limit device placement and cause device failure due to lead breakage or infection. Conventional wireless power delivery through biological tissue is difficult when devices are miniaturized and placed deep in the body. Here we show that magnetic materials can effectively harvest energy from magnetic fields and power millimeter-sized bioelectronics. These materials show excellent power densities even as the devices are made small allowing them to be fully implanted and wirelessly powered. We demonstrate that these mm-sized wireless devices can be used to power different types of conventional stimulation electrodes when implanted in rabbits, pigs, and freely moving rats. Furthermore, these miniature electrical stimulators can be adapted to power many individually addressable stimulation channels while still maintaining a small overall device footprint.

A fundamental goal of Neuroscience is to understand how the activity of specific neuronal circuits mediates behavior. Determining which aspects of neural activity are used by downstream circuits to guide behavior requires to manipulate activity while simultaneously monitoring behavioral readout. Holographic optical stimulation is an emerging toolbox for distributed control of spatiotemporal neuronal activity, which could shed new light on its direct link to behavior, by precisely manipulating the neural code while monitoring behavioral readout and neural responses.
I will describe new models, tools and experiments that together lead to recent advances in several fundamental technical aspects of optical neural interfacing and applications of such systems towards producing behaviorally relevant neural activity. First, I will focus on surprising new insights at the level of membrane biophysics that enable predictive modeling of both photo-thermal and optogenetic light-tissue effects. I will then describe our efforts to precisely probe neural circuits using rapid two-photon optogenetic stimulation and imaging with cellular resolution and ms-timescale temporal precision. Using high-energy lasers and rapid spatial light modulation we stimulate dozens of neurons deep (>250 µm) in brain tissue at a high rate. Our new system allows to generate and precisely manipulate artificial sensory responses. This approach has a broad range of potential applications in dissecting the activity codes that guide behavior across different modalities.

Recently, there is a growing interest in techniques for regulating the activity of nerve cells using light. The optical stimulation method has a higher spatiotemporal resolution than the electrical stimulation, and it is easy to implement multiple stimuli using multiple wavelengths. Although optogenetics, which is based on light-sensitive ion channels and pumps, has been widely used as an experimental tool owing to high cell specificity, the necessity of carrying out genetic manipulation is a limitation in terms of translation to clinical situation. To overcome this, nongenetic optical stimulation has been actively studied, and photothermal stimulation is one of them.
Photothermal stimulation is a method of modulating neuronal activity by applying heat to nerve cells via a substance that converts light into heat. Plasmmonic nanomaterials were used as transducers to induce photothermal effects. Among those nanomaterials, gold nanorods(GNRs) have been studied extensively in the field of nanomedicine on photothermal therapy because GNRs selectively absorb near infrared (NIR) rays and are highly efficient in converting light energy into thermal energy. Our group has been developing NIR sensitive GNR platform to suppress the neuronal activity, and investigated the inhibitory effect of photothermal stimulation on neuronal activity on in vitro neural activity.
In this presentation, I will present a GNR-based platform for locally applying photothermal stimulation to specific areas or cells of the network on a planar microelectrode array chip. In order to create a local heating pattern in tens of micrometer scale, two approaches were taken: patterning GNR particles onto the chip surface, or patterning the light. We applied inkjet printing technique to make high-quality GNR patterns in large area, and optimized the surface interaction with GNR ink droplets using a layer-by-layer technique. In order to pattern NIR light to the micrometer level, optical system was designed to achieve sufficient optical power density at a single cell size. We grew in vitro neural networks on microelectrode arrays to investigate the effect of local photothermal stimulation by multichannel spike recordings. When local photothermal stimulation was applied to a part of the network, the synchronized activity of the network was reduced and the degree of connectivity was changed. In the case of single cell-level photothermal stimulation, inhibition and rebound of action potential were observed. Comparing the optical power required for electrical activity inhibition, the action potential was instantaneously suppressed by using a very small level of optical power for single cell stimulation.
This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2018R1A2A1A05022604).

Although one of the most widespread approach to stimulate living systems relies on electrical stimuli, in recent years contactless and wireless methods are attracting a growing research interest. Within this context, light represents a clean and spatiotemporal precise tool to achieve effective bio-stimulation. With this in mind, conjugated molecules and macromolecules can be utilised as light-transducers and can offer an optimal platform to interface with cells and living organisms, given their relatively high optical absorption/emission cross sections, versatility in chemical synthesis and, in general, lower toxicity than their inorganic counterparts.
In this work, we focus on the light modulation of cardiomyocytes pacing. To achieve this, we investigate two interfacial configurations between the light transducer and the cells, namely a planar and a diffuse molecular one. For the planar architecture we make use of the well-known photoactive conjugated polymer poly(3-hexylthiophene), while for the molecular interface we employ a newly synthesized azobenzene photo-mechanical transducer.
The ultimate goal of this project is to produce an artificial photoresponsive smart tissue for future applications in robotic, pharmacological and regenerative medicine fields, as well as for tissue and neural regeneration.

Microalgae are extremely important photosynthetic organisms for both their environmental functions (e.g. CO₂ fixation and O₂ production) and industrial applications (e.g. food, drugs and bio-fuel production). In order to increase biomass production it is, indeed, of fundamental importance to increase the efficiency of microalgae photosynthesis. This approach fits perfectly into a circular economy scheme, in fact many waste products from industrial processes (e.g. CO₂, wastewater, sludge and agro-waste) can be used to feed photosynthetic processes to produce biomass. Among microalgae, diatoms constitute a major group that have found many industrial applications from biomedicine to photonics and they are particularly interesting for their hierarchically organized porous biosilica shells.
One possible strategy to enhance photosynthesis consists of filling the spectral gap in the light absorption of microalgae pigments (e.g. chlorophylls and carotenoids). The basic idea of this approach consists in introducing photoactive components which absorb light in the uncovered solar spectral regions and they transfer the collected energy to the photosynthetic units of the microalgae. Genetically engineered approaches can be exploited but, unfortunately, these tools in microalgae are still limited to few species.
In this work we demonstrate a new non-genetic approach to artificially increase the photosynthetic efficiency of Thalassiosira weissflogii diatoms by adding exogenous antennae cyanine molecules (Cy5) as a staining dye. In particular we observe that Cy5 is rapidly incorporated into the chloroplast of the microalgae without being toxic, increasing diatoms cell growth and photosynthetic oxygen production upon illumination. Cy5 dye was chosen as a possible FRET donor for chlorophyll a (acceptor) because of the following characteristics: (i) low toxicity, (ii) absorption/emission features and (iii) molecular structure.
By means of confocal microscopy we observe that Cy5 is localized in the shell and chloroplasts of the diatoms, where they can be still detected after 8 days. The incorporation of the dye is likely favored by the charged functional groups on the cyanine molecule which causes the electrostatic interaction between cyanine and cell membrane. Time-resolved fluorescence spectroscopy shows a decrease in the Cy5 excited state lifetime which is consistent with a resonant energy transfer (FRET) process between Cy5 (donor) and Chl a (acceptor). The quenching effect, and hence energy transfer, reaches a maximum after about 24 hours and, then, it gradually decreases never reaching the initial value after 6 days. In order to confirm the role of Cy5 as an enhancer of photosynthesis, photosynthetic activity was measured in the presence and absence of Cy5. The presence of Cy5 increases Pmax (maximum photosynthetic activity) by 49% compared to the control. In order to exclude that the increased productivity could be ascribed to any sort of Cy5 metabolism independent from light we repeated the measurement in the dark, observing no significant difference in cell density. This proves that only the simultaneous presence of Cy5 and light causes the diatoms growth increase. Finally it is worth stressing that the algae population is healthy for the whole experiment, and no toxic reactions or adverse effects in general have been observed.
In conclusion, in this work we demonstrated a novel method to increase in vivo photosynthetic activity in diatoms by adding Cy5 as exogenous antennae molecules. This route represents a convenient alternative to genetic modifications. This approach may be in principle extended to other photosynthetic organisms and it lays the foundations for the bio-photonic engineering of algal production by using photoactive molecules and it can impact on algae production technology, for instance extending the active volume of bioreactors and thus enhancing the yield.

Implantable nanophotonics devices and injectable nanomaterials with new functions interfacing with neural systems enables new techniques to fire neurons. High spatial resolution and deep penetration in brain provided by NIR light opens up potential to overcome fundamental limitation of other photo or magnetic driven neural modulation techniques. Specifically, we will report our recent progresses on photoacoustic devices and nanotransducers for successful in vitro and in vivo neuro stimulation. In the first application, we designed and developed a miniaturized fiber with the fiber tip with two layer coating: a diffusion layer and an expansion layer with nanocomposites to produce high intensity and controlled frequency of localized ultrasound when excited with nanosecond laser light. In vitro and in vivo neuron stimulation was successfully achieved with a laser duration of 20 millisecond. Neural stimulation in motor cortex of intact awaken mouse brains confirmed the photoacoustic stimulation was localized with a high spatial resolution of 500 microns and not through indirect auditory stimulation. Additionally, to enable non-invasive and MRI compatible neurostimulation, we developed a semiconducting polymer based nanotransducer, which generate ultrasound upon excitation of NIR II nanosecond laser light and perform stimulation when binding to neuron membrane. In vitro photoacoustic neuron stimulation with a successful rate of 80% is achieved with a laser duration of 3 millisecond. Different neuron response were demonstrated through controlling the binding between nanotransducers and neuron membrane.

Near Infrared light responsive photon upconversion nanoparticles possess a unique optical mechanism where long wavelength low-energy photons can be converted to high-energy emissions at the shorter wavelengths. They are highly promising for in vitro and in vivo optical imaging and therapy due to such special optical properties. Recently, upconversion nanoparticles (UCNPs) have stood out as a fascinating emerging tool in numerous biological and material applications. In this talk, I will first present new developments regarding engineering UCNPs towards wireless optogenetic applications in neuroscience and immunotherapy. Secondly, I will present our more recent development of an ocular injectable photoreceptor-binding upconversion nanoparticles that can create the mammalian infrared imaging vision in their naked eyes.

Direct control of cells homing to damaged myocardium after an ischemic insult is an overarching goal in the cardiac repair field because this will allow to prime the re-activation of the injured myocardium and vasculature.
Here we propose a novel strategy to gain optical control of Endothelial Progenitor Cell (EPC) fate, avoiding the drawbacks associated with current approaches, mainly based on cell therapy and electro-mechanical stimulation.
Our strategy is based on the combination of light sensitive conjugated polymers (CPs), used as photo-actuators, with the advantages offered by optical stimulation^1-4. At variance with cell therapy and electro-mechanical stimulation, light modulation offers unprecedented spatial and temporal resolution, permitting lower invasiveness and higher selectivity, and it allows to provide either excitation/inhibition of the cell activity. We focus on Endothelial Colony Forming Cells (ECFCs), which represent the only known EPCs subset truly belonging to the endothelial lineage showing robust in vitro proliferation and overwhelming vessel formation in vivo. We demonstrate that polymer-mediated optical excitation is able to induce a robust enhancement of cells proliferation and lumen formation in vitro. Moreover, we identify the pathways leading to this effective enhancement in ECFCs network formation, as due to light induced activation of transient receptor potential vanilloid channels⁵.
Altogether our results represent a novel effective application of semiconducting polymer-based optical modulation to induce angiogenesis in vitro. More in general, this work represents, to the best of our knowledge, the first report on use of CP for optical modulation of the cell fate, with important perspectives in cell-based therapy and regenerative medicine.

The restoration of light response with complex spatiotemporal features in retinal degenerative diseases towards retinal prosthesis has proven to be a considerable challenge over the past decades. Herein, inspired by the structure and function of photoreceptors in retinas, we develop artificial photoreceptors based on gold nanoparticle-decorated titania nanowire arrays, for restoration of visual responses in the blind mice with degenerated photoreceptors. Green, blue and near UV light responses in the retinal ganglion cells (RGCs) are restored with a spatial resolution better than 100 µm. ON responses in RGCs are blocked by glutamatergic antagonists, suggesting functional preservation of the remaining retinal circuits. Moreover, neurons in the primary visual cortex respond to light after subretinal implant of nanowire arrays. Improvement in pupillary light reflex suggests the behavioral recovery of light sensitivity. Our study will shed light on the development of a new generation of optoelectronic toolkits for subretinal prosthetic devices.

Hybrid interfaces composed by organic semiconductors in contact with living tissues are appealing for the controlled photo-stimulation of tissue cells in both in-vivo and in-vitro applications. Organic optoelectronic materials, such as light sensitive and conjugated polymers, provide an easy processable, biocompatible and tunable tool for the development of bio-devices and prosthesis able to restore vision. Planar organic prosthesis based on a thin film of poly(3-hexylthiophene) (P3HT) have been proven to be successful in inducing vision recovery in rat model affected by retinitis pigmentosa.
Considering such a promising result, we decided to investigate P3HT nanoparticles (NPs). According to preliminary results, P3HT NPs succeeded in restoring vision acuity in blind rats. However, despite the encouraging results, the mechanisms at the basis of the retinal organic prosthesis functioning is still not well understood.
In this work, we investigate the photophysical properties of light sensitive core-shell nanoparticle in which one component is P3HT. Different core materials were selected (among which: ITO, TiO₂ and Au) in order to have core-shell systems with different optoelectronic properties, permitting to investigate the cores effect on the organic NPs prosthesis.
Core-shell NPs provides a ground for investigating the photoexcitation mechanism and eventually controlling it by engineering ad hoc interfaces where energy level alignment can be predicted. NPs are a versatile system able to be widespread in the whole treated tissue. Moreover, NPS properties, such as tissue selectivity, light sensitivity, elasticity, etc. can be tuned, providing a versatile tool for the comprehension and improvement of the interaction between the prosthesis and the biological tissue.

The emerging understanding of cellular heterogeneity in complex biological systems has necessitated new tools for large-scale, single-cell analysis. Single-cell molecular analyses have led to identification of new cell types and discovery of novel targets for diagnosis and therapy. Despite these advances, a major challenge is the ability to tag and discriminate individual cells, and track them over time or over the course of different measurements. Current fluorescence-based approaches are fundamentally limited in multiplexing ability due to typical fluorophores emission linewidths, between 30-100 nm.
We have recently developed a novel class of imaging probes, called laser particles (LPs), with massive multiplexing capability and optimized properties for cell tagging applications. Our LPs are made of silica-coated semiconductor optical microcavities in a microdisk geometry, with ~2 μm diameter and 400 nm thickness. Upon optical pumping with a 1064 laser, the LPs generate single narrowband emission peaks (<0.5 nm) ranging from 1170 to 1580 nm, enabling massive spectral multiplexing. The LPs are readily internalized into cells to act as unique optical barcodes, where they occupy only ~0.1% of the cell volume.
In this contribution, I will present our results regarding the fabrication and characterization of these LPs, their stability and biocompatibility in biological environments and the use of these probes for wavelength-multiplexed cell tagging and imaging. I will demonstrate real-time tracking of thousands of individual cells in a 3D tumor spheroid invasion assay over several days showing different behavioral phenotypes.

The field of Optomechanics has made impressive advances in the last decades, covering a broad range of applications going from ultrasensitive sensing to fundamental quantum studies. The use of optomechanical devices for biosensing has acquired crescent interest in the last years, being semiconductor microdisks one of the most promising platform on this field.
Semiconductor microdisks support both, optical and mechanical modes, which possess extraordinary properties. Optically, semiconductor microdisks support the so called whispery gallery modes (WGMs), where the light circulates around the disk periphery. WGMs combine submycron optical mode volume and low optical dissipation, reaching optical quality factor above 10^5. From the mechanical point of view, they support a family of modes, the radial breathing modes (RBM), which present extremely high mechanical frequencies (> GHz) and low energy losses in liquids. These assets, together with their remarkably low masses (in the pg range), provide them with extremely low mass sensitivities and high speed, notably, while immersed in liquid [1]. Moreover, semiconductor microdisks can be integrated in collective configurations, thus, improving their sensing efficiency while keeping their individual capabilities [2].
Optomechanical microdisks allows to simultaneously monitor optical and mechanical modes, which provides them with access to the optical and mechanical properties of the given analyte. This dual sensing approach is fully innovative and significantly improves the sensor reliability and robustness. We have first applied this novel method on the detection of environmental changes, particularly temperature and humidity. We demonstrated that the method allows to decoupled humidity and temperature effects with extraordinary precision, which place them as excellent sensors for this purpose. Notably, in this work we show the first application of optomechanical devices as biological sensors. We have developed a novel deposition method which allow us to precisely locate individual, alive and intact bacteria in our sensors. By simultaneous detecting changes in their mechanical and optical modes, we probe that microdisks allows to determine the mechanical properties (rigidity, viscosity and mass) and the optical properties (index of refraction, optical absorption coefficient and size) of Staphylococcus Epidermidis bacteria.
Importantly, besides their capabilities to sense the analyte optical and mechanical properties, we also proven that optomechanical microdisks can detect the intrinsic mechanical resonances of the bacteria. This concept has been never realized even proposed before. By coupling the mechanical modes of the sensor with the ones of the analyte, the coupled system supports detectable collective mechanical modes, which provide precise information about the analyte modes. Importantly, coupling between the sensor and the analyte modes require having close resonance frequencies. It is important to note that the detection of analyte mechanical modes provides extremely high sensitivity on the determination of the morphology and mechanical properties of the analytes. In addition, the method allows to track in real-time the mechanical resonance frequency of the bacterium, which significantly increases the responsivity of the system to analyte changes. The proposed method radically departs from the classical paradigm for sensing the mass and stiffness of analytes with micromechanical resonators. This novel concept opens the door for studying biological interactions at the most fundamental level.

References
[1] Gil-Santos, E. et al. High-frequency nano-optomechanical disk resonators in liquids. Nature Nanotechnology, 2015. 10, p. 810-817.
[2] Gil-Santos, E. et al. Light-mediated cascaded locking of multiple nano-optomechanical oscillators. Physical Review Letters, 2017. 118, p. 063605.

We present how trans-membrane energy transfer and ionic interactions can be controlled across the bilayer membrane of large unilamellar vesicles. A system has been designed, where hydrophobic carbon dots (CDs) are embedded in the lipophilic part of lipid bilayers, while fluorescein molecules are covalently bound to the hydrophilic lipid heads [1]. The CDs are built of polycyclic aromatic hydrocarbon domains [2] that form biocompatible nanoparticles (~1.0 - 1.5 nm) with high photochemical stability. The spectral overlap between the CD emission and the fluorescein absorption spectra allows to establish trans-membrane Förster resonance energy transfer (FRET). We observe energy transfer efficiencies of 51% for a separation distance of ~4 nm between the donor CDs and the acceptor dyes, which is in good agreement with the bilayer geometry. Furthermore, we find that ionic interactions between the positively charged CDs and the anionic fluorescein let them conjoin within the vesicle membrane. This co-localization of both components does not only facilitate energy transfer, but also results in photoluminescence enhancement of the membrane bound dyes, which is beneficial for imaging applications and microscopy studies of membrane systems. Overall, this capability of balancing energy transfer and ionic interactions within a model membrane represents a general strategy to investigate trans-membrane related processes such as ion transport and changes of the membrane potential with high spatiotemporal control and resolution.

References:
[1] S.D. Pritzl, F. Pschunder, F. Ehrat, S. Bhattacharyya, T. Lohmüller, M. Huergo, J. Feldmann, Nano Letters, 2019.
[2] M. Fu, F. Ehrat, Y. Wang, K.Z. Milowska, C. Reckmeier, A.L. Rogach, J.K. Stolarczyk, A.S. Urban, J. Feldmann, Nano Letters, 15(9), p. 6030-6035, (2015).

The ability to interact with neuronal cells and to monitor their status plays a pivotal role in neuroscience, pharmacology and cell biology. In the last years, we deeply investigated both theoretically and experimentally the interactions of 3D nanostructured surface sensors with living cells such human neurons and cardiomyocytes [1]-[2]. The aim is to make an effective interface between the intracellular compartment and different class of nano-sensors including optical sensors, electrodes and nano-needles for intracellular delivery or sampling [3]. We developed a method based on plasmonic generation of nano-shockwaves [4] for opening transient nanopores into the cell membrane that is in close proximity with the plasmonic nanosensor. After the membrane poration the tip of the sensor is in direct contact with the intracellular compartment thus enabling intracellular investigations which include Raman traces of biomolecules [5]-[6], electrical recording of action potentials [7], and controlled delivery of single nanoparticles into selected cells [8]. However, for these applications have a real impact in the fields of Biology and Medicine it is necessary to make them available on the market, i.e. in the hands of biologist and researchers working in those fields. To this aim, we introduced the concept of planar meta-electrodes [9], namely a nanostructured surface that can work as electrode, a broad band plasmonic antenna, and optimal cellular interface. We show that meta-electrodes combined with commercial CMOS technology enable high quality intracellular electrical signals on the large network scale of human neuron and cardiomyocytes. Due to its robustness and easiness of use, we expect the method will be rapidly adopted by the scientific community and by pharmaceutical companies. In fact, the field suffers the lack of reliable approaches for pharmacological screening of drugs devoted to the central nervous system.
Also, we will take this opportunity to give a short overview of different types of optical and plasmonic biosensors we are currently developing. The latter includes single molecule Raman Sensors, DNA detection, and Protein sequencing.
References
[1] M. Dipalo et al., “Cells Adhering to 3D Vertical Nanostructures: Cell Membrane Reshaping without Stable Internalization”, Nano Letters, 18, 6100-6105, 2018.
[2] R. Capozza et al., “Cell Membrane Disruption by Vertical Micro-Nanopillars: Role of Membrane Bending and Traction Forces”,” ACS Applied Materials and Interfaces, 10, 29107-29114, 2018.
[3] A. Cerea at al., “Selective intracellular delivery and intracellular recordings combined in MEA biosensors”, LAB CHIP, 18, 3492, 2018.
[4] P. Zilio et al., “Hot electrons in water: injection and ponderomotive acceleration by means of plasmonic nanoelectrodes”, Light: Science & Applications 6 (6), e17002, 2017.
[5] V. Caprettini et al., “Enhanced Raman Investigation of Cell Membrane and Intracellular Compounds by 3D Plasmonic Nanoelectrode Arrays”, Advanced Science, 1800560, 2018.
[6] V. Shalabaeva et. al.,”Time resolved and label free monitoring of extracellular metabolites by surface enhanced Raman spectroscopy”, PloS one 12 (4), e0175581, 2017.
[7] M. Dipalo et al., “Intracellular and extracellular recording of spontaneous action potentials in mammalian neurons and cardiac cells with 3D plasmonic nanoelectrodes”. Nano Letters, 17, 3932-3939, 2017
[8] J. Huang et al., “Controlled Intracellular Delivery of Single Particles in Single Cells by 3D Hollow Nanoelectrodes”, Nano Letters, 19 (2), 722–731, 2019.
[9] M. Dipalo et al., “Plasmonic meta-electrodes allow intracellular recordings at network level on high-density CMOS-multi-electrode arrays”, Nature Nanotechnology 13, 965–971, 2018.

Innovations in light microscopy have revolutionized the way researchers study biological systems. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces several fundamental limitations such as the rather bulky fluorescent tags, color barrier for multiplex imaging, and limited ability for probing in vivo metabolism. To address these challenges, I will present three coherent Raman imaging strategies, respectively. First, we devised a Bioorthogonal Chemical Imaging platform suited for probing small bio-molecules. This scheme couples the emerging stimulated Raman scattering (SRS) microscopy with tiny vibrational probes. Exciting biomedical applications such as imaging fatty acid metabolism, glucose uptake and metabolism, drug trafficking, protein synthesis, DNA replication, and tumor metabolism will be presented. Second, we invented a super-multiplex optical imaging technique. We developed electronic pre-resonance SRS microscopy with high sensitivity. Chemically, we created vibrational palettes consisting of novel dyes. This super-multiplex optical imaging approach promises to facilitate untangling intricate interactions in complex biological systems, and can also find broad applications in photonics and biotechnology. Third, we introduced a platform that combines deuterium oxide (D2O) probing with SRS microscopy (DO-SRS) to image in situ metabolic activities in animals. Enzymatic incorporation of D2O-derived deuterium into macromolecules generates carbon-deuterium (C-D) bonds, which track biosynthesis in tissues in situ. Within the broad vibrational spectra of C-D bonds, we discovered lipid-, protein-, and DNA-specific Raman shifts with macromolecular selectivity. DO-SRS, being noninvasive and universally applicable, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.

Subcutaneous medical devices designed for cell therapy can be advanced by a prevascularization phase that proceeds the transplantation of tissue into the device. This approach holds promise to replace whole organ transplantation with thin vascularized devices hosting only the necessary supporting cells. We have developed a noninvasive optical technology to study the vascularization into such medical devices. In our technique, oxygen partial pressure within a device is monitored by Oxygen Sensitive Tubes (OSTs), comprising oxygen permeable silicone tubes coated on their inner surface with a porphyrin oxygen-sensitive dye. OSTs were placed within a PDMS device and transplanted into athymic nude mice. An optical probe placed on the skin excites the OSTs with a pulse of light and detects the luminescent lifetime of emitted light. The lifetime is uniquely related to oxygen partial pressure. Further, we developed a Dynamic Inhalation Gas Test (DIGT) to determine the oxygen transport rate between the microvasculature and the device. DIGT works by monitoring oxygen partial pressure during a step change in the inhaled gas oxygen content. We report changing DIGT oxygen dynamics from a series of experiments spanning eight weeks. Our study shows DIGT dynamics are unique to each implant, suggesting the important role of the host tissue response in the availability of oxygen over time.

Fluorescent sensors directly probe chemical kinetics in remote locations, transmitting the signal wirelessly to the observer. However, many biological organisms suffer from the limited penetration depth of light, obstructing the sensor use. Here, we present wavelength modulation spectroscopy (WMS) that improves the signal to noise ratio (SNR) in thick tissues by 100x. This allows the detection of near-infrared signals from sensors implanted at depths of up to 4.5 cm in chicken breast tissues. Additionally, WMS can eliminate the need of reference measurements, as it self-corrects for mechanical artefacts caused by tissue movements. WMS is enabled by the modulating the excitation wavelength across 50 nm in 0.2 s and measuring the changes in emission, which allows us to separate autofluorescent and fluorescent components of the response based on the period of modulation. We demonstrate the application of WMS on common near-infrared sensors, such as carbon nanotubes and ICG. We applied WMS to improve the SNR in sensing H2O2, riboflavin, and ascorbic acid in vivo. This technique may find a variety of uses in diagnostics, real-time therapeutics, and biochemical studies.

Rapid, accurate identification of pathogens and their antibiotic susceptibility/resistance are essential to improve patient prognosis, slow the spread of infectious diseases, contain epidemics, and mitigate the misuse of antibiotics. Unfortunately, the gold standard for bacterial identification and antibiotic susceptibility testing (AST) still rely on century-old culturing methods that typically takes few weeks to run. In this work, we introduce a novel platform that combines Raman scattering spectroscopy with machine-learning to enable the identification of individual pathogens solely through their Raman signatures. Notably, cellular membranes have a unique molecular composition that give rise to a characteristic Raman signature that can be used for cellular identification. However, practical implementation of Raman-based identification has been hindered by the reproducibility of Raman measurements at the single cell level. To overcome this challenge and allow for accurate single-cell classification using Raman spectroscopy, we developed a machine-learning classification algorithm. Particularly, we developed a convolutional neural network (CNN) trained on Raman spectra from individual pathogens. Our CNN architecture consists of 25 one-dimensional (1D) convolutional layers and residual connections - instead of two-dimensional images, it takes one-dimensional spectra as input. Our training dataset consists of 60,000 single-cell spectra from 30 bacterial and yeast isolates representing over 94% of all bacterial infections treated at Stanford Hospital. The training data we used was acquired at a rate of 1 s per individual cell, corresponding to a signal-to-noise ratio (SNR) of 4.1 - roughly an order of magnitude lower than typical reported bacterial spectra. Using this dataset, we show that the average pathogen strain-level classification accuracy achieved is 93.8%. When these strains are grouped based on their empiric antibiotic treatment, the classification accuracy increases to more than 99% for the treatment group classification. To further enable studying the changes in the Raman signatures of pathogens upon exposure to external stimuli such as antibiotics while improving the signal-to-noise ratio, we implemented a liquid chamber where mixtures of pathogens and gold nanorods were prepared. Using a palette of gold nanorods with resonances ranging from 650nm to 920nm and a 785nm excitation laser, we show that significant enhancement in the Raman signatures of both E. Coli (Gram-negative) and Staphylococcus (Gram-positive) can be achieved. Interestingly, the obtained enhancements are achieved using two different nanorod sizes for the two tested species; while nanorods with shorter resonant wavelengths result in a more pronounced enhancement for Gram-negative E. Coli, longer-wavelength resonant nanorods produce the largest enhancement for the Gram-positive Staphylococcus. This is due to the tendency of nanorod aggregation with the Gram-negative E. Coli, due to the bacterial and nanorod surface charge interaction, causing the nanorod frequencies to red-shift. Our results lay the foundation for a fast, all-optical pathogen identification and antibiotic susceptibility classification beyond the conventional slow culture methods.

Photonic crystals (PhCs) have been largely employed as detection/sensing devices in recent years, since the photonic stop-band can be tuned by applying a number of external stimuli, such as chemical¹, thermal² and mechanical triggers³. In this context, we have recently proposed porous 1D photonic structures exhibiting electro-optical tunability, due to the incorporation of optoelectronically-active plasmonic nanoparticles in the photonic structures.^4–6
Here, we show that a hybrid plasmonic/photonic crystal consisting of a thin layer of bioactive plasmonic material (i.e. silver) deposited on top a 1D PhC can detect one of the most common bacterial contaminant, namely Escherichia coli. We speculate that the change in the plasmon charge density brought about by metal/bacterium interaction results in a variation of the plasmon resonance which, in turns, translates in a shift of the photonic structural color.

References
¹ W. Hong, X. Hu, B. Zhao, F. Zhang, and D. Zhang, Adv. Mater. 22, 5043 (2010).
² T. Kanai, D. Lee, H.C. Shum, R.K. Shah, and D.A. Weitz, Adv. Mater. 22, 4998 (2010).
³ H. Fudouzi and T. Sawada, Langmuir 22, 1365 (2006).
⁴ E. Aluicio-Sarduy, S. Callegari, D.G.F. del Valle, A. Desii, I. Kriegel, and F. Scotognella, Beilstein J. Nanotechnol. 7, 1404 (2016).
⁵ G.M. Paternò, C. Iseppon, A. D’Altri, C. Fasanotti, G. Merati, M. Randi, A. Desii, E.A.A. Pogna, D. Viola, G. Cerullo, F. Scotognella, and I. Kriegel, Sci. Rep. 8, 3517 (2018).
⁶ G.M. Paternò, L. Moscardi, I. Kriegel, F. Scotognella, and G. Lanzani, J. Photonics Energy 8, 1 (2018).

Studying the uptake and transport dynamics of engineered nanomaterials (ENMs) is an important step in designing next-generation drug delivery systems and in understanding the environmental impact of existing materials. However, normalizing transport across multiple particles and cells can be difficult due to differences in uptake time and cellular morphologies. Additionally, ENM transport is innately coupled with cell motility and membrane dynamics, making it challenging to draw quantitative comparisons between particle trajectories. Current studies also often depend on surface-bound fluorescent labels, which have the potential to alternative cellular recognition events. As a result, there is still a need to develop methods capable of monitoring ENM-cell interactions in a quantitative fashion, independent of specific surface modifications. Addressing these concerns, here we show how scatter enhanced phase contrast (SEPC) microscopy, a dual light source microscopy technique, can serve as a generalized label-free approach for monitoring nanoparticle uptake and transport dynamics. By avoiding fluorescent labels, SEPC allows for a rational exploration of the surface properties of nanomaterials in their native state. We demonstrate this experimentally, showing that SEPC works for a variety of metal and metal oxides, including Au, Ag, TiO2, CeO2, Al2O3, and Fe2O3 nanoparticles. Additionally, we show that when combined with microcontact printing, a technique which can be used to control cell morphology, we are able to normalize dynamics across multiple cells, enabling a quantitative study of ensemble nanoparticle uptake (n= ~480 particle trajectories). Using this approach, we observed that particles began as evenly distributed across the cell surface but became clustered in the perinuclear region after a short duration, indicating the bulk of ENM transport occurs within the first 30 minutes of exposure. These studies also suggested further spatial heterogeneities in ENM dynamics, revealing three distinct regions of particle transport across the cell, with particles moving faster near the edge of the cell and slowing down as they approach the nucleus. Overall, this indicated that membrane dynamics play an important role in regulating initial particle flow, while intracellular vesicular transport dictates intermediate dynamics. Collectively, this ensemble approach to studying transport dynamics has important implications for designing next-generation ENM-based drug delivery systems and in better understanding the uptake of existing materials.

The wound-healing assay is one of the most useful methods for the study of collective cell migration and cell-to-cell interaction. Furthermore, it provides an inexpensive and easy tool for therapeutics, by observing the effect of various chemical treatments on wound healing speed. Numerous studies have observed some novel mechanical and molecular interactions between the cells during the healing process using this assay.
Nonetheless, most are remaining at the stage of two-dimensional analysis such as gap closure rate, direct counting of cells or individual cell tracking, although it clearly involves a complex three-dimensional (3D) process. There are limitations when viewing the healing mechanism in 2-dimension. People do not have a full understanding of collective cell migration yet. The 3-dimensional (3D) properties of cells like thickness or velocity vector during the process present a new perspective to understand how cells near and away from the boundary migrate and interact with the neighboring cells. Here, we utilized non-labeling 3D long-term imaging and developed analysis tools for the wound healing assay.
Optical diffraction tomography (ODT) is an effective tool for imaging low-scattering samples like cells by utilizing intrinsic physical information in the sample without labeling. Based on laser interferometry, it reconstructs a three-dimensional refractive index tomogram of live cells or tissue, from multiple 2D measurements of optical field images of the sample illuminated from various angles. ODT provides label-free and quantitative phase imaging capability. Especially in terms of the experimental complexity or availability, its label-free imaging ability without difficulties such as photobleaching has an incomparable strength in monitoring the healing process for an extended period of time. A wide enough field of view for analysis can be covered by using a stitching algorithm. Thus, ODT with the aid of the stitching algorithm is an excellent fit for imaging the expanded area of wound healing model for an extended period of time in 3D.
To investigate dynamic 3D motions of cells in a wound-healing assay, we have systematically controlled the ODT imaging system and a motorized sample stage in a synchronized manner. We have measured a large area of a sample (up to 1 mm x 1 mm) with a high resolution (down to 110 nm and 360 nm in the lateral and axial spatial resolution, respectively) at a high frame rate (up to 3-5 tomograms per second for a specific field of view 81 μm x 81 μm).
We studied both the overall shape and the subcellular structures of individual cells in a time-lapse manner. Several quantifiable 3D properties such as the thickness of cells or condensation level of the nuclei chromatin in groups of cells with different chemical treatments are explored by employing the system. Especially, an examination of RI distribution in the nucleus of cells revealed different chromatin condensation level near the boundary and the inner regions of cells. This provides a new tool to study and monitor the wound healing mechanism and how the chemical treatments affect cells.
Our 3D non-labeling imaging and analyzing techniques of wound healing will deliver a significant impact on the pharmaceutical industry. Quantitative 3D analysis for migrating cells’ behavior during the wound healing allows a unique chance to study and analyze with precision the wound healing mechanisms. Studying more complex 3D wound healing models utilizing this technique will be the subsequent step which may give us a fundamental understanding of the in-vivo wound healing process.