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RESUMO O presente artigo configura-se como um estudo teórico-conceitual que integra duas pesquisas desenvolvidas, respectivamente, em nível de doutorado e de pós-doutorado em Educação. O texto tem por objetivo compreender a inclusão e o letramento digital do idoso, a partir da perspectiva da educação ao longo da vida, fundamentada na acepção humanista do conceito. O exponencial avanço tecnológico e o crescimento da população de pessoas idosas no Brasil e no mundo tornam pungente a necessidade de discutir a inclusão digital dessa faixa etária, bem como a ideia de uma educação que perdure durante todo o processo de viver. A inclusão digital impacta diretamente o pleno exercício da cidadania dos sujeitos sociais contemporâneos, uma vez que as linguagens hipermidiáticas do ciberespaço permeiam várias práticas sociais. Nesse contexto, o artigo propõe uma discussão entre quatro conceitos, a saber: aprendizagem ao longo da vida, inclusão digital, letramento digital e empoderamento (na acepção freiriana do termo). Considera-se que a inclusão digital é uma das facetas da inclusão social do idoso, pelo seu relevante papel na conquista da sua autonomia e do pleno exercício da cidadania. Os conceitos ora apresentados – letramento digital do idoso, aprendizagem ao longo da vida e empoderamento – buscam fornecer argumentos conceituais para melhor compreender as possíveis contribuições da inclusão digital, em uma sociedade marcada pelo fortalecimento das linguagens hipermidiáticas junto às práticas sociais contemporâneas. Palavras-chave: Idoso. Inclusão Digital. Letramento Digital. Educação ao Longa da Vida. Empoderamento. ABSTRACT This article is configured as a theoretical-conceptual study, which integrates two researchers developed at the doctoral and post-doctoral level in Education. The text aims to understand digital literacy from the perspective of lifelong education, based on the humanistic perspective of the concept. The exponential technological advance and the growth of the population of elderly people in Brazil and in the world make the need to discuss the digital inclusion of this age group, as well as the idea of an education that lasts throughout the process of living. Digital inclusion directly impacts the full exercise of citizenship by contemporary social subjects, since the hypermedia languages of cyberspace permeate a lot of social practices. In this context, the article seeks discussion between four concepts: lifelong learning, digital inclusion, digital literacy and empowerment (in the freiriana sense of the term). It is considered that digital inclusion is understood as one of the facets of the social inclusion of the elderly, due to its relevant role, in the achievement of their autonomy and their full exercise of citizenship. The concepts presented here - digital literacy of the elderly, lifelong learning and empowerment - seek to provide conceptual arguments to better understand the possible contributions of digital inclusion, in a society marked by the strengthening of hypermedia languages along with contemporary social practices. Keywords: Elderly. Digital inclusion. Digital Literacy. Lifelong Education. Empowerment.

RESUMO O presente artigo configura-se, em termos metodológicos, como um estudo teórico-conceitual, que integra uma pesquisa desenvolvida em nível de pós-doutoramento em Educação. O texto tem por objetivo desenvolver reflexões acerca da importância da inclusão digital para as pessoas com 60 anos ou mais. O exponencial avanço tecnológico e o crescimento da população de pessoas idosas no Brasil tornam pungente a necessidade de discutir a inclusão digital dessa faixa etária. A inclusão digital impacta diretamente o pleno exercício da cidadania dos sujeitos sociais contemporâneos, uma vez que as linguagens hipermidiáticas do ciberespaço permeiam várias práticas sociais. Nesse contexto, o artigo busca aproximações entre três conceitos, a saber: aprendizagem ao longo da vida, inclusão digital, empoderamento (na acepção freireana do termo). Considera-se que a inclusão digital é entendida como uma das facetas da inclusão social do idoso, pelo relevante papel, na conquista da sua autonomia e do seu pleno exercício da cidadania. A tríade conceitual ora apresentada – inclusão digital do idoso, aprendizagem ao longo da vida, empoderamento – busca fornecer argumentos conceituais para melhor compreender as possíveis contribuições da inclusão digital da pessoa idosa em uma sociedade marcada pelo fortalecimento das linguagens hipermidiáticas junto às práticas sociais contemporâneas. Palavras-chaves: Linguagens hipermidiáticas. Inclusão Digital do idoso. Empoderamento. Aprendizagem ao longo da vida. Autonomia. ABSTRACT This article is configured, in methodological terms, as a theoretical-conceptual study, which is part of a research developed at the post-doctoral level in Education. The article reflects on the importance of digital inclusion for people aged 60 or over. The exponential technological advance and the growth of the population of elderly people in Brazil make the need to discuss the digital inclusion of this age group. Digital inclusion directly impacts the full exercise of citizenship by contemporary social subjects, since the hypermedia languages of cyberspace permeate various social practices. In this context, this article seeks approximations among three concepts: lifelong learning, digital inclusion, empowerment (in a Freirean perspective). It is considered that digital inclusion is one of the faces of the social inclusion of the elderly, due to its relevant role in the achievement of their autonomy and their full exercise of citizenship. The conceptual triad presented here - digital inclusion of the elderly, lifelong learning, empowerment - seeks to provide conceptual arguments to better understand the possible contributions of digital inclusion for the elderly, in a society marked by the strengthening of hypermedia languages in the contemporary social practices. Keywords: Hypermedia languages. Digital inclusion of the elderly. Empowerment. Lifelong learning. Autonomy.

Este artigo aborda as interações vivenciadas por alunos em uma comunidade de prática online criada para dar sustentação a um curso no formato e-Learning, para funcionários da área de logística do Serviço Social do Comércio do Estado de São Paulo (Sesc SP). Trata-se de um estudo de caso etnográfico, cujo objetivo foi investigar o papel do fórum de discussões online tendo como fio condutor reflexões oriundas da teoria da complexidade, por meio de conceitos como autopoiese, auto-eco-organização e circularidade, usados como fundamentos explicativos dos aspectos observados. Os resultados mostram a utilização da educação online como meio de criar redes que podem contribuir para o fortalecimento de uma cidadania planetária, uma vez que explora uma proposta pedagógica ativa, dialógica e interativa, capaz de fomentar malhas solidárias de cooperação e a promoção de ambientes polissêmicos, que são favorecidos pelas reflexões advindas de problemas emergentes e suas possíveis soluções que podem ser instigadas por meio do protagonismo na formação.

Pensar o desenvolvimento de um curso no formato eletrônico na perspectiva do pensamento complexo de Edgar Morin remete à busca pela compreensão da transdisciplinaridade e como ela se apresenta desafiadora e inovadora para as reflexões acerca de assuntos que fazem parte de ambientes educacionais corporativos. Este artigo pretende elucidar o conceito da transdisciplinaridade que se apresenta em três pilares: a complexidade, a lógica do terceiro incluído, e os diferentes níveis de realidade. Esta pesquisa visou a analisar, à luz dessa teoria, as experiências desenvolvidas no SESC-SP com funcionários que participaram de curso de formação no formato e-learning associado a fórum de discussão. Os dados analisados foram coletados no ano de 2015, e a análise considerou os princípios da transdisciplinaridade tendo como ênfase as interações realizadas em ambiente virtual de aprendizagem. Os resultados demonstraram expressivos índices de concordância dos alunos em relação às questões apresentadas, o que nos permite reconhecer nessa metodologia de ensino/aprendizagem princípios intimamente relacionados com as questões transdisciplinares.

Abstract: Chronic pain associated with osteoarthritis (OA) remains an intractable problem with few effective treatment options. New approaches are needed to model the disease biology and to drive discovery of therapeutics. We present an in vitro model of OA pain, where dorsal root ganglion (DRG) sensory neurons were sensitized by a defined mixture of disease-relevant inflammatory mediators, here called Sensitizing PAin Reagent Composition or SPARC. Osteoarthritis-SPARC components showed synergistic or additive effects when applied in combination and induced pain phenotypes in vivo. To measure the effect of OA-SPARC on neural firing in a scalable format, we used a custom system for high throughput all-optical electrophysiology. This system enabled light-based membrane voltage recordings from hundreds of neurons in parallel with single cell and single action potential resolution and a throughput of up to 500,000 neurons per day. A computational framework was developed to construct a multiparameter OA-SPARC neuronal phenotype and to quantitatively assess phenotype reversal by candidate pharmacology. We screened ∼3000 approved drugs and mechanistically focused compounds, yielding data from over 1.2 million individual neurons with detailed assessment of functional OA-SPARC phenotype rescue and orthogonal "off-target" effects. Analysis of confirmed hits revealed diverse potential analgesic mechanisms including ion channel modulators and other mechanisms including MEK inhibitors and tyrosine kinase modulators. Our results suggest that the Raf-MEK-ERK axis in DRG neurons may integrate the inputs from multiple upstream inflammatory mediators found in osteoarthritis patient joints, and MAPK pathway activation in DRG neurons may contribute to chronic pain in patients with osteoarthritis., (Copyright © 2023 International Association for the Study of Pain.)

Video-based screening of pooled libraries is a powerful approach for directed evolution of biosensors because it enables selection along multiple dimensions simultaneously from large libraries. Here we develop a screening platform, Photopick, which achieves precise phenotype-activated photoselection over a large field of view (2.3 × 2.3 mm, containing >10 3  cells, per shot). We used the Photopick platform to evolve archaerhodopsin-derived genetically encoded voltage indicators (GEVIs) with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). These GEVIs gave improved signals in cultured neurons and in live mouse brains. By combining targeted in vivo optogenetic stimulation with high-precision voltage imaging, we characterized inhibitory synaptic coupling between individual cortical NDNF (neuron-derived neurotrophic factor) interneurons, and excitatory electrical synapses between individual hippocampal parvalbumin neurons. The QuasAr6 GEVIs are powerful tools for all-optical electrophysiology and the Photopick approach could be adapted to evolve a broad range of biosensors., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Optogenetic assays provide a flexible, scalable, and information rich approach to probe compound effects for ion channel drug targets in both heterologous expression systems and associated disease relevant cell types. Despite the potential utility and growing adoption of optogenetics, there remains a critical need for compatible platform technologies with the speed, sensitivity, and throughput to enable their application to broader drug screening applications. To address this challenge, we developed the Swarm TM , a custom designed optical instrument for highly parallelized, multicolor measurements in excitable cells, simultaneously recording changes in voltage and calcium activities at high temporal resolution under optical stimulation. The compact design featuring high power LEDs, large numerical aperture optics, and fast photodiode detection enables all-optical individual well readout of 24-wells simultaneously from multi-well plates while maintaining sufficient temporal resolution to probe millisecond response dynamics. The Swarm delivers variable intensity blue-light optogenetic stimulation to enable membrane depolarization and red or lime-light excitation to enable fluorescence detection of the resulting changes in membrane potential or calcium levels, respectively. The Swarm can screen ~10,000 wells/day in 384-well format, probing complex pharmacological interactions via a wide array of stimulation protocols. To evaluate the Swarm screening system, we optimized a series of heterologous optogenetic spiking HEK293 cell assays for several voltage-gated sodium channel subtypes including Nav1.2, Nav1.5, and Nav1.7. The Swarm was able to record pseudo-action potentials stably across all 24 objectives and provided pharmacological characterization of diverse sodium channel blockers. We performed a Nav1.7 screen of 200,000 small molecules in a 384-well plate format with all 560 plates reaching a Z' > 0.5. As a demonstration of the versatility of the Swarm, we also developed an assay measuring cardiac action potential and calcium waveform properties simultaneously under paced conditions using human induced pluripotent stem (iPS) cell-derived cardiomyocytes as an additional counter screen for cardiac toxicity. In summary, the Swarm is a novel high-throughput all-optical system capable of collecting information-dense data from optogenetic assays in both heterologous and iPS cell-derived models, which can be leveraged to drive diverse therapeutic discovery programs for nervous system disorders and other disease areas involving excitable cells., Competing Interests: GB, HZ, BH, JJ, JG, RC, DZ, AB, CW, YL, SN, OM, and GD are current or former employees of Q-State Biosciences and may hold stock options in Q-State Biosciences., (Copyright © 2022 Borja, Zhang, Harwood, Jacques, Grooms, Chantre, Zhang, Barnett, Werley, Lu, Nagle, McManus and Dempsey.)

Optogenetics provides a powerful approach for investigating neuronal electrophysiology at the scale required for drug discovery applications. Probing synaptic function with high throughput using optogenetics requires robust tools that enable both precise stimulation of and facile readout of synaptic activity. Here we describe two functional assays to achieve this end: (1) a pre-synaptic calcium assay that utilizes the channelrhodopsin, CheRiff, patterned optogenetic stimulus, and the pre-synaptically targeted calcium reporter jRGECO1a to monitor pre-synaptic changes in calcium influx and (2) a synaptic transmission assay in which CheRiff and cytosolic jRGECO1a are expressed in non-overlapping sets of neurons, enabling pre-synaptic stimulation and post-synaptic readout of activity. This chapter describes the methodology and practical considerations for implementation of these two assays.

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.

The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.

Induced pluripotent stem (iPS) cells offer the exciting opportunity for modeling neurological disorders in vitro in the context of a human genetic background. While significant progress has been made in advancing the use of iPS cell-based disease models, there remains an unmet need to characterize the electrophysiological profile of individual neurons with sufficient throughput to enable statistically robust assessment of disease phenotypes and pharmacological modulation. Here, we describe the Optopatch platform technology that utilizes optogenetics to both stimulate and record action potentials (APs) from human iPS cell-derived excitatory neurons with similar information content to manual patch clamp electrophysiology, but with ~  3 orders of magnitude greater throughput. Cortical excitatory neurons were produced using the NGN2 transcriptional programming approach and cultured in the presence of rodent glial cells. Characterization of the neuronal preparations using immunocytochemistry and qRT-PCR assays reveals an enrichment of neuronal and glutamatergic markers as well as select ion channels. We demonstrate the scale of our intrinsic cellular excitability assay using pharmacological assessment with select ion channel modulators quinidine and retigabine, by measuring changes in both spike timing and waveform properties. The Optopatch platform in human iPS cell-derived cortical excitatory neurons has the potential for detailed phenotype and pharmacology evaluation, which can serve as the basis of cellular disease model exploration for drug discovery and phenotypic screening efforts.

Transmembrane voltage and intracellular calcium concentration are coupled parameters essential to the function of neurons, cardiomyocytes, and other excitable cells. Here we introduce the Firefly-HR microscope for simultaneous optogenetic stimulation and voltage and calcium imaging with fluorescent proteins using three spectrally distinct visible color bands. Firefly-HR combines patterned stimulation, near-total internal reflection laser excitation through a prism located between the sample and a water-immersion objective, and concurrent imaging of three color channels. The microscope has efficient light collection, low fluorescent background, and a large field of view (0.24 x 1.2 mm @ 1000 frames/sec). We characterize optical crosstalk and demonstrate capabilities with three applications: (1) probing synaptically connected neuronal microcircuits, (2) examining the coupling between neuronal action potentials and calcium influx, and (3) studying the pharmacology of paced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) via simultaneous recordings of voltage, calcium, and contraction., Competing Interests: CAW: Q-State Biosciences (I,E,P), All other authors: Q-State Biosciences (I,E).

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our 'Firefly' microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology ('Optopatch') in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes., Competing Interests: CAW: Harvard University (P), Q-State Biosciences (I,E). AEC: Harvard University (P), Q-State Biosciences (I,C).

A key challenge for establishing a phenotypic screen for neuronal excitability is measurement of membrane potential changes with high throughput and accuracy. Most approaches for probing excitability rely on low-throughput, invasive methods or lack cell-specific information. These limitations stimulated the development of novel strategies for characterizing the electrical properties of cultured neurons. Among these was the development of optogenetic technologies (Optopatch) that allow for stimulation and recording of membrane voltage signals from cultured neurons with single-cell sensitivity and millisecond temporal resolution. Neuronal activity is elicited using blue light activation of the channelrhodopsin variant 'CheRiff'. Action potentials and synaptic signals are measured with 'QuasAr', a rapid and sensitive voltage-indicating protein with near-infrared fluorescence that scales proportionately with transmembrane potential. This integrated technology of optical stimulation and recording of electrical signals enables investigation of neuronal electrical function with unprecedented scale and precision. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation.

There is a pressing need for new and more effective treatments for central nervous system (CNS) disorders. A large body of evidence now suggests that alterations in synaptic transmission and neuronal excitability represent underlying factors for many neurological and psychiatric diseases. However, it has been challenging to target these complex functional domains for therapeutic discovery using traditional neuronal assay methods. Here we review advances in neuronal screening technologies and cellular model systems that enable phenotypic screening of neuronal function as a basis for novel CNS drug discovery approaches., (Copyright © 2017. Published by Elsevier Ltd.)

Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

We explored the use of the optically transparent semiconductor indium tin oxide (ITO) as an alternative to optically opaque metals for the fabrication of photonic structures in terahertz (THz) near-field studies. Using the polaritonics platform, we confirmed the ability to clearly image both bound and leaky electric fields underneath an ITO layer. We observed good agreement between measured waveguide dispersion and analytical theory of an asymmetric metal-clad planar waveguide with TE and TM polarizations. Further characterization of the ITO revealed that even moderately conductive samples provided sufficiently high quality factors for studying guided and leaky wave behaviors in individual transparent THz resonant structures such as antennas or split ring resonators. However, without higher conductive ITO, the limited reflection efficiency and high radiation damping measured here both diminish the applicability of ITO for high-reflecting, arrayed, or long path-length elements.

Multidimensional spectroscopy at visible and infrared frequencies has opened a window into the transfer of energy and quantum coherences at ultrafast time scales. For these measurements to be performed in a manageable amount of time, one spectral axis is typically recorded in a single laser shot. An analogous rapid-scanning capability for THz measurements will unlock the multidimensional toolkit in this frequency range. Here, we first review the merits of existing single-shot THz schemes and discuss their potential in multidimensional THz spectroscopy. We then introduce improved experimental designs and noise suppression techniques for the two most promising methods: frequency-to-time encoding with linear spectral interferometry and angle-to-time encoding with dual echelons. Both methods, each using electro-optic detection in the linear regime, were able to reproduce the THz temporal waveform acquired with a traditional scanning delay line. Although spectral interferometry had mediocre performance in terms of signal-to-noise, the dual echelon method was easily implemented and achieved the same level of signal-to-noise as the scanning delay line in only 4.5% of the laser pulses otherwise required (or 22 times faster). This reduction in acquisition time will compress day-long scans to hours and hence provides a practical technique for multidimensional THz measurements.

Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The "photostick" technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns.

Genetically encoded fluorescent reporters of membrane potential promise to reveal aspects of neural function not detectable by other means. We present a palette of multicoloured brightly fluorescent genetically encoded voltage indicators with sensitivities from 8-13% ΔF/F per 100 mV, and half-maximal response times from 4-7 ms. A fluorescent protein is fused to an archaerhodopsin-derived voltage sensor. Voltage-induced shifts in the absorption spectrum of the rhodopsin lead to voltage-dependent nonradiative quenching of the appended fluorescent protein. Through a library screen, we identify linkers and fluorescent protein combinations that report neuronal action potentials in cultured rat hippocampal neurons with a single-trial signal-to-noise ratio from 7 to 9 in a 1 kHz imaging bandwidth at modest illumination intensity. The freedom to choose a voltage indicator from an array of colours facilitates multicolour voltage imaging, as well as combination with other optical reporters and optogenetic actuators.

All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators.

We investigate the interaction between terahertz waves and resonant antennas with sub-cycle temporal and λ/100 spatial resolution. Depositing antennas on a LiNbO₃ waveguide enables non-invasive electro-optic imaging, quantitative field characterization, and direct measurement of field enhancement (up to 40-fold). The spectral response is determined over a bandwidth spanning from DC across multiple resonances, and distinct behavior is observed in the near- and far-field. The scaling of enhancement and resonant frequency with gap size and antenna length agrees well with simulations.

A photodiode and data acquisition card whose sampling clock is synchronized to the repetition rate of a laser are used to measure the energy of each laser pulse. Simple analysis of the data yields the noise spectrum from very low frequencies up to half the repetition rate and quantifies the pulse energy distribution. When two photodiodes for balanced detection are used in combination with an optical modulator, the technique is capable of detecting very weak pump-probe signals (ΔI/I(0) ~ 10(-5) at 1 kHz), with a sensitivity that is competitive with a lock-in amplifier. Detection with the data acquisition card is versatile and offers many advantages including full quantification of noise during each stage of signal processing, arbitrary digital filtering in silico after data collection is complete, direct readout of percent signal modulation, and easy adaptation for fast scanning of delay between pump and probe.

Femtosecond optical pulses were used to generate THz-frequency phonon polariton waves in a 50 micrometer lithium niobate slab, which acts as a subwavelength, anisotropic planar waveguide. The spatial and temporal electric field profiles of the THz waves were recorded for different propagation directions using a polarization gating imaging system, and experimental dispersion curves were determined via a two-dimensional Fourier transform. Dispersion relations for an anisotropic slab waveguide were derived via analytical analysis and found to be in excellent agreement with all observed experimental modes. From the dispersion relations, we analyze the propagation-direction-dependent behavior, effective refractive index values, and generation efficiencies for THz-frequency modes in the subwavelength, anisotropic slab waveguide.

We apply phase contrast imaging to enable, sharply focused visualization of terahertz waves in electro-optic media. The approach allows quantitative characterization of THz waves in the 60 GHz - 4.5 THz frequency range in a thin dielectric slab and in-focus observation of THz waves in polaritonic structures.

Thin, platelike single crystals of p-terphenyl (PT) doped with terrylene impurity molecules can be prepared by spin-coating from solution. Strikingly, individual terrylene molecules can be observed traveling inside the crystal over distances of several micrometers by using single-molecule fluorescence imaging at room temperature. Analysis of the motion by single-particle tracking and correlation methods indicates that the molecules act as nanoprobes by exploring long, thin crack-like defects with correlated orientations, defects that can be difficult to observe by other means. Apparently, the regions accessible to the moving molecules are in the interior of the crystal and hence are partially protected from oxidation. In addition to the traveling molecules, which photobleach in times on the order of 32 s under continuous irradiation at 2 kW/cm2, two other spatially fixed populations are observed: one with transition dipole oriented along the c-axis of the crystal with a characteristic photobleaching time greater than 32 h, and one with a characteristic photobleaching time of 18 min.