Neurotransmission between neurons, which can occur over the span of a few milliseconds, relies on the controlled release of small molecule neurotransmitters, many of which are amino acids. Fluorescence imaging provides the necessary speed to follow these events and has emerged as a powerful technique for investigating neurotransmission. In this review, we highlight some of the roles of the 20 canonical amino acids, GABA and β-alanine in neurotransmission. We also discuss available fluorescence-based probes for amino acids that have been shown to be compatible for live cell imaging, namely those based on synthetic dyes, nanostructures (quantum dots and nanotubes), and genetically encoded components. We aim to provide tool developers with information that may guide future engineering efforts and tool users with information regarding existing indicators to facilitate studies of amino acid dynamics.
Ratiometric photoluminescent detection of the toxicologically potent organophosphate ester nerve agents paraoxon (PX) and parathion (PT) using the complementary optical and chemical properties of the long Stokes shift green fluorescent protein variant, mAmetrine1.2 (mAm), and redemitting silicon-based quantum dots (SiQDs) is reported. PX and PT selectively quench SiQD photoluminescence (PL) through a dynamic quenching mechanism, thereby, facilitating the development of a ratiometric sensor platform that shows micromolar limits of detection for PX and PT and that is unaffected by the presence of common inorganic and organic interferents. As a part of the present study, we also demonstrate that the paper-based sensors derived from mAm and SiQDs detect PX and PT at concentrations as low as 5 μM using a readily available commercial color analysis smartphone "app". The ratiometric sensor reported herein can potentially be used for the convenient and rapid on-site detection and quantification of PX and PT in real-world samples.
Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca2+ dynamics and neuronal activities on a single cell level. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized these far-red GECIs as purified proteins and assessed their performance when expressed in cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644 nm, respectively, display large responses to Ca2+ (ΔF/F0 = 6 for FR-GECO1a, 18 for FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs also have high affinities (apparent Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca2+, and they enable sensitive and fast detection of single action potentials in neurons.
The brain relies on many forms of dynamic activities in individual neurons, from synaptic transmission to electrical activity and intracellular signaling events. Monitoring these neuronal activities with high spatiotemporal resolution in the context of animal behavior is a necessary step to achieve a mechanistic understanding of brain function. With the rapid development and dissemination of highly optimized genetically encoded fluorescent sensors, a growing number of brain activities can now be visualized in vivo. To date, cellular calcium imaging, which has been largely used as a proxy for electrical activity, has become a mainstay in systems neuroscience. While challenges remain, voltage imaging of neural populations is now possible. In addition, it is becoming increasingly practical to image over half a dozen neurotransmitters, as well as certain intracellular signaling and metabolic activities. These new capabilities enable neuroscientists to test previously unattainable hypotheses and questions. This review summarizes recent progress in the development and delivery of genetically encoded fluorescent sensors, and highlights example applications in the context of in vivo imaging.
To address the issue of poor selectivity in nanotechnologydriven, portable nitroaromatic sensors, we have coupled a ratiometric photoluminescent sensor based on silicon quantum dots and fluorescent proteins with a colorimetric, enzyme-based sensor. Together, the sensors allow differentiation of nitroaromatic compounds�specifically, distinguishing acetylcholinergic nerve agents from the explosive compounds explored herein. The combined system can detect 2,4,6-trinitrotoluene, 2,4dinitrotoluene, and 4-nitrophenol with micromolar detection limits and affords subsequent differentiation from the nitro-containing nerve agent paraoxon. A prototype portable sensing device housing both technologies is presented and tested for achieving visual differentiation of the nitroaromatic explosives versus nerve agents. This demonstrates the advantage of merging elements of materials chemistry and biochemistry to devise customized sensors that can accurately identify hazardous chemical species.
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