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Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration
摘要： Potassium ion (K+) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K+ indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are F?rster resonance energy transfer (FRET)-based indicators with a bacterial K+ binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K+ imaging) is a single fluorescent protein-based K+ indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K+ at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K+ depletion in live cells and K+ efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca2+ indicator, enable dual-color imaging of K+ and Ca2+ dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K+ dynamics.
关键词： FRET-based sensors,potassium ion imaging,single fluorescent protein sensors,intracellular K+ dynamics,genetically encoded indicators
更新于2025-11-21 11:24:58
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A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice
摘要： A bright, red-shifted Genetically Encoded Voltage Indicator (GEVI) was developed using a modified version of the fluorescent protein, tdTomato. Dimerization of the fluorescent domain for ArcLight-type GEVIs has been shown to affect the signal size of the voltage-dependent optical signal. For red-shifted GEVI development, tdTomato was split fusing a single dTomato chromophore to the voltage sensing domain. Optimization of the amino acid length and charge composition of the linker region between the voltage sensing domain and the fluorescent protein resulted in a probe that is an order of magnitude brighter than FlicR1 at a resting potential of ?70 mV and exhibits a ten-fold larger change in fluorescence (ΔF) upon 100 mV depolarization of the plasma membrane in HEK 293 cells. Unlike ArcLight, the introduction of charged residues to the exterior of dTomato did not substantially improve the dynamic range of the optical signal. As a result, this new GEVI, Ilmol, yields a 3-fold improvement in the signal-to-noise ratio compared to FlicR1 despite a smaller fractional change in fluorescence of 4% per 100 mV depolarization of the plasma membrane. Ilmol expresses well in neurons resolving action potentials in neuronal cultures and reporting population signals in mouse hippocampal acute brain slice recordings. Ilmol is the brightest red-shifted GEVI to date enabling imaging with 160-fold less light than Archon1 for primary neuron recordings (50 mW/cm2 versus 8 W/cm2) and 600-fold less light than QuasAr2 for mouse brain slice recordings (500 mW/cm2 versus 300 W/cm2). This new GEVI uses a distinct mechanism from other approaches, opening an alternate engineering path to improve sensitivity and speed.
关键词： fluorescence,Genetically Encoded Voltage Indicator,tdTomato,optical signal,GEVI,neuronal activity,red-shifted,voltage imaging
更新于2025-09-23 15:21:01
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Genetically encoded FRET-based optical sensor for Hg2+ detection and intracellular imaging in living cells
摘要： Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (Kd) of 5.09 × 10?7 M, thus providing a detection range for Hg2+ quantification between 0.210?μM and 1.196?μM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intra- cellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.
关键词： Genetically encoded,FRET,Fluorescent proteins,Mercury,Nanosensors
更新于2025-09-11 14:15:04
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Intravitreal AAV-Delivery of Genetically Encoded Sensors Enabling Simultaneous Two-Photon Imaging and Electrophysiology of Optic Nerve Axons
摘要： Myelination of axons by oligodendrocytes is a key feature of the remarkably fast operating CNS. Oligodendrocytes not only tune axonal conduction speed but are also suggested to maintain long-term axonal integrity by providing metabolic support to the axons they ensheath. However, how myelinating oligodendrocytes impact axonal energy homeostasis remains poorly understood and difficult to investigate. Here, we provide a method of how to study electrically active myelinated axons expressing genetically encoded sensors by combining electrophysiology and two-photon imaging of acutely isolated optic nerves. We show that intravitreal adeno-associated viral (AAV) vector delivery is an efficient tool to achieve functional sensor expression in optic nerve axons, which is demonstrated by measuring axonal ATP dynamics following AAV-mediated sensor expression. This novel approach allows for fast expression of any optical sensor of interest to be studied in optic nerve axons without the need to go through the laborious process of producing new transgenic mouse lines. Viral-mediated biosensor expression in myelinated axons and the subsequent combination of nerve recordings and sensor imaging outlines a powerful method to investigate oligodendroglial support functions and to further interrogate cellular mechanisms governing axonal energy homeostasis under physiological and pathological conditions.
关键词： genetically encoded sensors,myelinated axons,intravitreal AAV injection,ATP-sensor ATeam1.03YEMK,optic nerve recording,two-photon imaging
更新于2025-09-10 09:29:36
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In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish
摘要： Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.
关键词： sensory neuroscience,Issue 141,calcium imaging,GCaMP,Neuroscience,genetically encoded indicators,hair cells,in vivo imaging,Zebrafish,lateral line,confocal imaging
更新于2025-09-04 15:30:14
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Microscopy of the Heart || Optogenetic Tools in the Microscopy of Cardiac Excitation-Contraction Coupling
摘要： Microscopy became a scienti?c investigation method in the seventeenth century with the application of the ?rst build microscopes on biological samples [1, 2]. Soon it became a popular method to stain samples in order to visualise particular (cellular and subcellular) structures [3]. These stains, either based on absorption or ?uorescence, have limitations in respect to their speci?city and are often toxic to cells, which limits investigations to short intervals or even dead samples. In 1987 the idea came up to use a ?uorescent protein that was discovered 25 years before [4], in particular a green ?uorescent protein (GFP) form the medusa Aequorea victoria to label cells and cellular structures [5]. With the sequencing and cloning of GFP, a so-called ‘green revolution’ started, which led to regular usage of ?uorescent proteins as markers or sensors (for details see below) in the majority of cellular research in physiology, microbiology, pharmacology, molecular biology, anatomy, cell biology, biophysics and many other biomedical ?elds. Although the expression of the ?uorescent proteins and their optical investigation can already be regarded as optogenetic tools, this term was only applied when the optical properties of proteins were used to manipulate cells. The best-known example of such a protein is the channelrhodopsin, a light-gated ion channel [6, 7]. When this ion channel is expressed in a membrane and illuminated with light of the appropriate wavelength, the channel will be activated and opened, which results in passive transportation of ions across the membrane and a change of the membrane potential. However, within this chapter we consider both aspects, the observation and the manipulation as optogenetic tools. To use the optogenetic tool, the genes of these proteins need to be transferred into the cells to allow the expression of the protein. For an overview of gene delivery into target cells, see [8].
关键词： channelrhodopsins,cardiac excitation-contraction coupling,F?rster Resonance Energy Transfer,genetically encoded biosensors,Optogenetic tools,microscopy
更新于2025-09-04 15:30:14