All cells maintain ionic gradients across their plasma membranes, producing transmembrane potentials (Vmem). Mounting evidence suggests a relationship between resting Vmem and the physiology of non-excitable cells with implications in diverse areas, including cancer, cellular differentiation, and body patterning. A lack of non-invasive methods to record absolute Vmem limits our understanding of this fundamental signal. To address this need, we developed a fluorescence lifetime-based approach (VF-FLIM) to visualize and optically quantify Vmem with single-cell resolution in mammalian cell culture. Using VF-FLIM, we report Vmem distributions over thousands of cells, a 100-fold improvement relative to electrophysiological approaches. In human carcinoma cells, we visualize the voltage response to growth factor stimulation, stably recording a 10–15 mV hyperpolarization over minutes. Using pharmacological inhibitors, we identify the source of the hyperpolarization as the Ca2+-activated K+ channel KCa3.1. The ability to optically quantify absolute Vmem with cellular resolution will allow a re-examination of its signaling roles.

Microbial redox activity offers a potentially transformative approach to the low-temperature synthesis of nanostructured inorganic materials. Diverse strains of the dissimilatory metal-reducing bacteria Shewanella are known to produce photoactive filamentous arsenic sulfide nanomaterials by reducing arsenate and thiosulfate in anaerobic culture conditions. Here we report in situ microscopic observations and measure the thermally activated (79 kJ mol(-1)) precipitation kinetics of high yield (504 mg per liter of culture, 82% of theoretical maximum) extracellular As2S3 nanofibers produced by Shewanella sp. strain ANA-3, and demonstrate their potential in functional devices by constructing field effect transistors (FETs) based on individual nanofibers. The use of strain ANA-3, which possesses both respiratory and detoxification arsenic reductases, resulted in significantly faster nanofiber synthesis than other strains previously tested, mutants of ANA-3 deficient in arsenic reduction, and when compared to abiotic arsenic sulfide precipitation from As(III) and S(2-). Detailed characterization by electron microscopy, energy-dispersive X-ray spectroscopy, electron probe microanalysis and Tauc analysis of UV-vis spectrophotometry showed the biogenic precipitate to consist primarily of amorphous As2S3 nanofibers with an indirect optical band gap of 2.37 eV. X-ray diffraction also revealed the presence of crystalline As8S(9-x) minerals that, until recently, were thought to form only at higher temperatures and under hydrothermal conditions. The nanoscale FETs enabled a detailed characterization of the charge mobility (∼10(-5) cm(2) V(-1) s(-1)) and gating behavior of the heterogeneously doped nanofibers. These studies indicate that the biotransformation of metalloids and chalcogens by bacteria enables fast, efficient, sustainable synthesis of technologically relevant chalcogenides for potential electronic and optoelectronic applications.

Voltage imaging with fluorescent indicators offers a powerful complement to traditional electrode or Ca2+-imaging approaches for monitoring electrical activity.

Near infrared (NIR) fluorophores may hold the key for non-invasive optical imaging of deep structures in intact organisms with high spatial and temporal resolution. Yet, developing fluorescent dyes that emit and absorb light at wavelengths greater than 700 nm and that respond to biochemical and biophysical events in living systems remains an outstanding challenge. Here, we report the design, synthesis, and application of NIR-absorbing and -emitting, sulfonated, phosphine-oxide (po) rhodamines for voltage imaging in thick tissue from the central nervous system. We find po-rhodamine based voltage reporters, or poRhoVRs, display NIR excitation and emission profiles at greater than 700 nm, show best-in class voltage sensitivity (up to 43% ΔF/F per 100 mV in HEK cells), and can be combined with existing optical sensors, like Ca 2+ -sensitive fluorescent proteins (GCaMP), and actuators, like light-activated opsins ChannelRhodopsin-2 (ChR2). Simultaneous voltage and Ca 2+ imaging reveals differences in activity dynamics in rat hippocampal neurons, and pairing poRhoVR with blue-light based ChR2 affords all-optical electrophysiology. In ex vivo retinas isolated from a mouse model of retinal degeneration, poRhoVR, together with GCaMP-based Ca 2+ imaging and traditional multi-electrode array (MEA) recording, can provide a comprehensive physiological activity profile of neuronal activity. Taken together, these experiments establish that poRhoVR will open new horizons in optical interrogation of cellular and neuronal physiology in intact systems.

Recent advances in the design and application of redox-active fluorescent proteins (FPs) stimulated an interest in the electronic structure of the ionized/electron-detached FP chromophores. Here, we report the results of a computational study of the electron-detached and ionized states of model chromophores of green and red FPs. We focus on the analysis of the effects of the phenolate OH group position (ortho, meta, and para) on relative energies of the chromophores in the ground as well as in the ionized/detached electronic states. We found that, similarly to the green chromophore, the red chromophores with the OH group in meta position have lower vertical detachment energies (DE) and greater ionization energies rela-tive to the ortho and para forms. Moreover, the effect is stronger for the red anionic chromophores. The differences in DE in meta species relative to their para counterparts are 0.47 and 0.25 eV for the red and green chromophores, respectively. The observed trends are due to a combined effect of resonance stabilization and the electronegativity of the acylimine group in the red chromophores. The analysis is supported by the computed charge and spin density delocalization patterns. V C