Name: imaging membrane potential with two types of genetically encoded fluorescent voltage sensors
Uploaded: 2016-02-04T14:00:00
Description: Watch this Scientific Journal Video about Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors at JoVE.com

This work was supported by the World Class Institute (WCI) program of the National Research Foundation of Korea funded by Ministry of Education, Science, and Technology of Korea Grant WCI 2009-003 and Korea Institute of Science and Technology Institutional Program Project 2E24210. Sungmoo Lee was supported by Global Ph.D. Fellowship program (NRF-2013H1A2A1033344) of the National Research Foundation (NRF) under the Ministry of Education (MOE, Korea).

dichiarazione etica: Il protocollo sperimentazione animale è stato approvato dalla cura degli animali e del Comitato Istituzionale Usa presso KIST protocollo animale 2014-001. 1. Installazione apparecchiature <ol><li> configurazione Imaging <ol><li> Inserire un microscopio invertito a fluorescenza su un tavolo isolamento dalle vibrazioni. Utilizzare un alto ingrandimento (lente immersione in olio 60X con 1,35 apertura numerica) lente obiettivo e un cubo filtrante con uno specchio dicroico...

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Il sistema nervoso utilizza tensione in diversi modi, inibizione provoca un leggero iperpolarizzazione, ingresso sinaptica provoca un leggero depolarizzazione e un&#39;azione potenziali risultati in un cambiamento relativamente grande tensione. La possibilità di misurare le variazioni del potenziale di membrana dalla Gevis offre il potenziale promettente di analizzare diversi componenti di circuiti neuronali contemporaneamente. In questo rapporto si dimostra un metodo fondamentale per le modifiche di imaging del potenz...

Il principale obiettivo di questo lavoro è quello di dimostrare l&#39;imaging ottico delle variazioni di potenziali di membrana in vitro utilizzando proteine ​​fluorescenti geneticamente codificate. cambiamenti di imaging del potenziale di membrana offre l&#39;eccitante possibilità di studiare l&#39;attività dei circuiti neuronali. Quando i cambiamenti nella membrana risultato potenziale in un cambiamento intensità di fluorescenza, ogni pixel della telecamera diventa un elettrodo surrogata per misure non intrusivi di attività neuronale. Da oltre quarant&#39;anni, coloranti organici voltaggio-sensibile sono stati utili per osservare i cambiamenti nel potenziale di membrana 1-4. Tuttavia, questi coloranti mancano di specificità cellulare. Inoltre, alcuni tipi di cellule sono difficili da macchiare. indicatori di tensione geneticamente codificati (GeViS) superare queste limitazioni avendo le cellule da studiare specificamente esprimono la sonda fluorescente tensione-sensibili. Ci sono tre classi di Gevis. La prima classe di GEVI utilizza il vodominio della fosfatasi tensione-sensing ensione-sensing con una singola proteina fluorescente (FP) 5-9 o un paio 10-12 un Förster trasferimento di energia di risonanza (FRET). La seconda classe di sensori utilizza microbica rodopsina come indicatore fluorescente direttamente 13-15 o tramite FRET elettrocromico 16,17. La terza classe utilizza due componenti, il componente genetica essendo un FP membrana ancorata ed un secondo componente essendo una membrana vincolato tempra colorante 18-20. Mentre il secondo e terzo classi sono utili per esperimenti in vitro e fetta 19,20, solo la prima classe di sensori sono attualmente utile per analisi in vivo 6. In questo rapporto ci sarà dimostrare l&#39;imaging del potenziale di membrana utilizzando la prima classe di Gevis (figura 1) in vitro. Questa prima classe di sensori di tensione è il più facile da passare a imaging in vivo. Dal momento che Gevis utilizing un dominio di rilevamento della tensione fuso ad una FP sono circa 50 volte più luminoso classe rodopsina dei sensori, possono essere ripreso con illuminazione lampada ad arco anziché richiedere un laser estremamente potente. Un&#39;altra conseguenza della disparità di luminosità è che la prima classe di GeViS può facilmente superare l&#39;auto-fluorescenza del cervello. Le sonde rodopsina-based non può. La terza classe di sensore è altrettanto chiaro come la prima classe, ma richiede l&#39;aggiunta di un quencher chimico che è difficile da gestire in vivo. Noi, quindi, dimostrare l&#39;acquisizione di una sonda con un singolo FP (Bongwoori) 8 e una sonda costituita da una coppia FRET (Nabi 2) 12. Il FRET costruisce in questo rapporto sono versioni farfalla di VSFP-CR (proteine ​​fluorescenti voltaggio-sensibile - Clover-mRuby2) 11 costituito da un donatore fluorescente verde, trifoglio, e un accettore fluorescente rossa, mRuby2, chiamato Nabi 2.242 e 2.244 Nabi <sup&gt; 12. L&#39;introduzione di questi tipi di registrazioni dovrebbe dare ai ricercatori una migliore comprensione del tipo di Gevis di informazione in grado di fornire. <img alt="Figura 1" src="/files/ftp_upload/53566/53566fig1.jpg" /> Figura 1. due tipi di indicatori geneticamente codificati tensione (Gevis) Divisori stampati in questo rapporto (A) Un FP mono basato GEVI avere un dominio di tensione-sensing trans-membrana e una proteina fluorescente. (B) Un GEVI basato FRET composto da un dominio di tensione-sensing trans-membrana, un donatore tasto e accettore. <a href="https://www.jove.com/files/ftp_upload/53566/53566fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

<table border="0" cellpadding="0" cellspacing="0" width="923">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Inverted Microscope</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">IX71</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">60X objective lens (numerical aperture = 1.35)</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">UPLSAPO 60XO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Excitation filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">&#160;FF02-472/30</td>
			<td rowspan="3" style="width:187px;">For voltage imaging of super ecliptic pHluorin in Bongwoori</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF495-Di03-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF01-497/LP</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">75W Xenon arc lamp</td>
			<td style="width:165px;">CAIRN</td>
			<td style="width:203px;">OptoSource Illuminator</td>
			<td style="width:187px;">LEDs and lasers are also effective light sources</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Slow speed CCD camera</td>
			<td style="width:165px;">Hitachi</td>
			<td style="width:203px;">KP-D20BU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dual port camera adaptor</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">U-DPCAD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">High speed CCD camera</td>
			<td style="width:165px;">RedShirtImaging, LLC</td>
			<td style="width:203px;">NeuroCCD-SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Image splitter</td>
			<td style="width:165px;">CAIRN</td>
			<td style="width:203px;">Optosplit 2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Excitation filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF01-475/23-25</td>
			<td rowspan="5" style="width:187px;">For voltage imaging of FRET pair based GEVI consisting of Clover and mRuby2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF495-Di03-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Chroma</td>
			<td style="width:203px;">ET520/40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF560-FDi01-25X36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Chroma</td>
			<td style="width:203px;">ET645/75</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Vibration isolation system</td>
			<td style="width:165px;">Kinetic systems</td>
			<td style="width:203px;">250BM-IC, 5702E-3036-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Patching chamber</td>
			<td style="width:165px;">Warner instruments</td>
			<td style="width:203px;">RC-26G, 64-0235</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">#0 Micro Coverglass (22x40mm)</td>
			<td style="width:165px;">Electron Microscopy Sciences</td>
			<td style="width:203px;">72198-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Temperature controller</td>
			<td style="width:165px;">Warner instruments</td>
			<td style="width:203px;">TC-344B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">#0 (0.08~0.13mm) - 10mm diameter glass coverslip</td>
			<td style="width:165px;">Ted Pella</td>
			<td style="width:203px;">260366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Lipofection agent</td>
			<td style="width:165px;">Life Technologies</td>
			<td style="width:203px;">11668-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Calcium phosphate reagent</td>
			<td style="width:165px;">Clontech - Takara</td>
			<td style="width:203px;">631312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Patch clamp amplifier</td>
			<td style="width:165px;">HEKA</td>
			<td style="width:203px;">EPC 10 USB amplifier</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Multi-channel data acquisition software</td>
			<td style="width:165px;">HEKA</td>
			<td style="width:203px;">Patchmaster</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Image acquisition and analysis software</td>
			<td style="width:165px;">RedShirtImaging</td>
			<td style="width:203px;">Neuroplex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Spreadsheet application software</td>
			<td style="width:165px;">Microsoft</td>
			<td style="width:203px;">Microsoft Excel 2010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Data analysis software</td>
			<td style="width:165px;">OriginLab</td>
			<td style="width:203px;">OriginPro 8.6.0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Demagnifier</td>
			<td style="width:165px;">Qioptiq LINOS</td>
			<td style="width:203px;">Optem standard camera coupler 0.38x SC38 J clamp</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Confocal microscope</td>
			<td style="width:165px;">Nikon</td>
			<td style="width:203px;">Nikon A1R confocal microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Anti-fade reagent</td>
			<td style="width:165px;">Life Technologies</td>
			<td style="width:203px;">P36930</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging membrane potential with two types of genetically encoded fluorescent voltage sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

Transitoriamente cellule transfettate possono presentare una significativa variazione di intensità di fluorescenza e il grado di espressione membrana plasmatica. Anche sullo stesso vetrino alcune cellule avranno diversi livelli di fluorescenza interna. Questo è probabilmente dovuto alla quantità di agente trasfezione assorbita dalla cella. Di tanto in tanto, troppo espressione fa sì che la cellula di sperimentare la risposta proteina spiegato con conseguente apoptosi 27 (l...

Watch this Scientific Journal Video about Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors at JoVE.com

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Imaging potenziale di membrana con due tipi di geneticamente codificati fluorescenti di sensori di tensione

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Research

JoVE Journal

Neuroscience

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Imaging ottico di neuroni nel ganglio Granchio Stomatogastric con tensione sensibili Coloranti

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Ricerca

Neuroscienze

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Determinazione della mitocondriale Oxygen Species potenziale di membrana e reattiva in neuroni corticali di ratto dal vivo

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

Le risposte di imaging neuronale in preparazione Fetta di organo vomeronasale Esprimere un sensore di calcio geneticamente codificato

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

Cella di misura singola di rilascio di dopamina con simultanea di tensione-clamp e Amperometria

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Potenziale di membrana Dye Imaging di ventromediale dell&#39;ipotalamo neuroni da topi adulti per studiare glucosio Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Imaging intracellulare Ca<sup&gt; 2+</sup&gt; Segnali di striatale astrociti da topi adulti utilizzando indicatori di calcio geneticamente codificate

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Controllo ottico di una proteina neuronale Utilizzando un acido Innaturale Amino geneticamente codificata in neuroni

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

Imaging Dell'assorbimento mitocondriale di Ca2+ in astrociti e neuroni utilizzando indicatori Ca2+ geneticamente codificati (GECI)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...