A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure ...membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.
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•In vivo, two-photon imaging of novel genetically encoded voltage indicators•Observing sensory stimulus-evoked voltage signals with subcellular resolution•Calcium signals, unlike voltage signals, are compartmentalized within a neuron•ON and OFF selectivity arises in the transformation between voltage and calcium
Observation of subcellular changes in membrane potential and calcium concentration using two-photon imaging of genetically encoded indicators illuminates neuronal computations in vivo, including the origin of ON and OFF selectivity.
Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic ...range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Monomeric red and far-red FPs and indicators now perform nearly as well as the best green FPs (and indicators).
Reversible and irreversible photochromism in FPs can be exploited to increase optical resolution and improve contrast compared with traditional fluorescence microscopy.
Infrared FPs (IFPs) are becoming ever more useful as labels for various proteins that allow correct localization and whole-animal imaging. IFPs can serve as an additional fluorescent ‘color’ for simultaneous imaging with visible FP-labeled proteins.
Bacterial phytochrome (BphP)-based IFPs provide a new scaffold for engineering fluorogenic indicators, which are ideal to visualize spatiotemporal dynamics of cell signaling in vivo.
Small ultra-red FP (smURFP) is the brightest far-red nonprototypical FP (comparable with EGFP) and is extremely photostable. smURFP may prove particularly useful as a photostable FP for super-resolution imaging and as a FRET acceptor for biosensing applications.
The engineering of new fluorescent indicators that combine features of prototypical FP-based indicators with photochromic proteins can reveal the cellular maps of biochemical activities in super-resolution.
FPs can be used as optogenetic actuators to manipulate cellular and protein functions through chromophore-assisted light inactivation or light-controlled protein oligomerization.
Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural ...photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.
A Guide to Fluorescent Protein FRET Pairs Bajar, Bryce T; Wang, Emily S; Zhang, Shu ...
Sensors (Basel, Switzerland),
09/2016, Letnik:
16, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high ...spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.
Nanodiamond (ND) has been proposed for various biomedical applications, including bioimaging, biosensing and drug delivery, owing to its physical-chemical properties and biocompatibility. ...Particularly, ND has been demonstrated as fluorescence- and Raman-detectable labels in many cellular models. Different surface functionalization methods have been developed, varying the ND's surface properties and rendering the possibility to attach biomolecules to provide interaction with biological targets. For this, toxicity is of major concern in animal models. Aside from cellular models, a cost-effective animal test will greatly facilitate the development of applications. In this study, we use the rapid, sensitive and reproducible zebrafish embryo model for in vivo nanotoxicity test. We optimize the conditions for using this animal model and analyze the zebrafish embryonic development in the presence of ND. ND is observed in the embryo in vivo using laser confocal fluorescence microscopy and fluorescence lifetime imaging. Using the zebrafish model for a safety evaluation of ND-based nanolabel is discussed.
As one of the most important engineering materials, aluminum alloys have been widely applied in many fields. However, the requirement of enhancing their mechanical properties without sacrificing the ...ductility is always a challenge in the development of aluminum alloys. Thanks to the excellent physical and mechanical properties, graphene nanoflakes (GNFs) have been applied as promising reinforcing elements in various engineering materials, including polymers and ceramics. However, the investigation of GNFs as reinforcement phase in metals or alloys, especially in aluminum alloys, is still very limited. In this study, the aluminum alloy reinforced by GNFs was successfully prepared via powder metallurgy approach. The GNFs were mixed with aluminum alloy powders through ball milling and followed by hot isostatic pressing. The green body was then hot extruded to obtain the final GNFs reinforced aluminum alloy nanocomposite. The scanning electron microscopy and transmission electron microscope analysis show that GNFs were well dispersed in the aluminum alloy matrix and no chemical reactions were observed at the interfaces between the GNFs and aluminum alloy matrix. The mechanical properties׳ testing results show that with increasing filling content of GNFs, both tensile and yield strengths were remarkably increased without losing the ductility performance. These results not only provided a pathway to achieve the goal of preparing high strength aluminum alloys with excellent ductilitybut they also shed light on the development of other metal alloys reinforced by GNFs.
The Leicester Cough Questionnaire (LCQ) has been validated for assessing cough-specific health status in bronchiectasis. We translated the LCQ into Mandarin Chinese and investigated its validity, ...reliability and responsiveness.
The LCQ was translated into Mandarin Chinese using the forward-backward translation procedure. A total of 144 out-patients completed the Mandarin Chinese version of the LCQ (LCQ-MC), the Hospital Anxiety and Depression Scale (HADS) and the St George's Respiratory Questionnaire. Reassessments were performed during exacerbations and at 6 months. Concurrent validation, internal consistency, repeatability and responsiveness were determined.
Minor cultural adaptations were made to the wording of LCQ-MC. No other difficulties were found during the translation process, with all items easily adapted to acceptable Mandarin Chinese. The questionnaire was not changed in terms of content layout and the order of the questions. In cognitive debriefing interviews, participants reported that the questionnaire was acceptable, relevant, comprehensive and easy to complete. The LCQ-MC showed good concurrent validity, internal consistency and test-retest reliability. Responsiveness was shown by significant changes in LCQ-MC scores between steady state, the first exacerbation and following 2-week antibiotic treatment (both interval changes, P < 0.01) CONCLUSION: The LCQ-MC is a valid, reliable and responsive instrument for determining cough-specific health status in Chinese bronchiectasis patients.
ABSTRACT
The closest stars that harbor potentially habitable planets are cool M-stars. Upcoming ground- and space-based telescopes will be able to search the atmosphere of such planets for a range of ...chemicals. To facilitate this search and to inform upcoming observations, we model the high-resolution reflection spectra of two of the closest potentially habitable exoplanets for a range of terrestrial atmospheres and surface pressures for active and inactive phases of their host stars for both oxic and anoxic conditions: Proxima b, the closest potentially habitable exoplanet, and Trappist-1e, one of 3 Earth-size planets orbiting in the Habitable Zone of Trappist-1. We find that atmospheric spectral features, including biosignatures like O2 in combination with a reduced gas like CH4 for oxic atmospheres, as well as climate indicators like CO2 and H2O for all atmospheres, show absorption features in the spectra of Proxima b and Trappist-1e models. However for some features like oxygen, high-resolution observations will be critical to identify them in a planet's reflected flux. Thus these two planets will be among the best targets for upcoming observations of potential Earth-like planets in reflected light with planned Extremely Large Telescopes.
Protein kinases transduce signals to regulate a wide array of cellular functions in eukaryotes. A generalizable method for optical control of kinases would enable fine spatiotemporal interrogation or ...manipulation of these various functions. We report the design and application of single-chain cofactor-free kinases with photoswitchable activity. We engineered a dimeric protein, pdDronpa, that dissociates in cyan light and reassociates in violet light. Attaching two pdDronpa domains at rationally selected locations in the kinase domain, we created the photoswitchable kinases psRaf1, psMEK1, psMEK2, and psCDK5. Using these photoswitchable kinases, we established an all-optical cell-based assay for screening inhibitors, uncovered a direct and rapid inhibitory feedback loop from ERK to MEK1, and mediated developmental changes and synaptic vesicle transport in vivo using light.
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