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).

Etikk uttalelse: The dyreforsøk protokollen ble godkjent av Institutional Animal Care og bruk komité ved Kist dyr protokollen 2014-001. 1. Oppsett Utstyr <ol><li> Imaging oppsett <ol><li> Plasser en omvendt fluorescens mikroskop på en vibrasjonsisolering bord. Bruke en høy forstørrelse (60x olje nedsenking linse med 1,35 numerisk apertur) objektiv linse og et filter kube utstyrt med et dikroisk speil og filtre egnet for de fluorescerende proteiner som brukes for spennings avbildning. ...

<ol>
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Nervesystemet benytter spenning på flere forskjellige måter, fører til hemming en svak hyperpolarisering, fører synaptisk inngangs en svak depolarisering og et aksjonspotensial resulterer i en forholdsvis stor spenningsendring. Evnen til å måle endringer i membranpotensial ved GEVIs tilbyr det lovende potensialet til å analysere flere komponenter av nevronale kretser samtidig. I denne rapporten demonstrerer vi en grunnleggende metode for avbildning forandringer i membranpotensialet ved hjelp av GEVIs. <p clas...

De viktigste fokus for denne artikkelen er å vise den optiske bilde av endringer i membranpotensialer in vitro ved bruk av genetisk kodet fluorescerende proteiner. Imaging endringer i membranpotensiale tilbyr spennende mulighet til å studere aktiviteten til nevrale kretser. Når forandringer i membranpotensialet resulterer i en fluorescens-intensitet endring, blir hver piksel i kameraet et surrogat elektrode som gjør det mulig nonintrusive målinger av neuronal aktivitet. I over førti år har organiske spenningsfølsomme fargestoffer vært nyttig for å observere endringer i membranpotensialet 1-4. Men disse fargestoffer mangler cellulær spesifisitet. I tillegg er noen celletyper er vanskelige å farge. Genetisk kodede spenningsindikatorer (GEVIs) overvinne disse begrensningene ved at cellene som skal studeres spesifikt uttrykker det fluorescerende spenningsfølsom sonde. Det er tre klasser av GEVIs. Den første klassen av Gevi bruker voltage-sensing domenet fra spenningsføler fosfatase med enten en enkel fluorescerende protein (FP) 5-9 eller en Förster resonans energi overføring (FRET) pair 10-12. Den andre klasse av sensorer benytter mikrobiell rhodopsin som en fluorescerende indikator direkte 13-15 eller via elektrokromatiske FRET 16,17. Den tredje klasse utnytter to komponenter, den genetiske komponenten være en membran forankret FP og en andre komponent er et membranbundet bråkjøling fargestoff 18-20. Mens den andre og tredje klasser er nyttige for in vitro-forsøk og eksperimenter stykke 19,20, bare den første klasse av sensorer er for tiden nyttige for in vivo-analyser 6. I denne rapporten vil vi demonstrere avbildning av membranpotensialet ved bruk av første klasse av GEVIs (figur 1) in vitro. Denne første klasse av spenningssensorene er den enkleste å gå over til in vivo imaging. Siden GEVIs utilizing en spenningsføler domene fusert til et FP er omtrent 50-ganger sterkere enn den rhodopsin klasse av sensorer, de kan avbildes ved hjelp av buelampe belysning i stedet for å kreve en ekstremt kraftig laser. En annen konsekvens av den forskjellen i lysstyrke er at den første klassen av GEVIs lett kan overstige auto-fluorescens av hjernen. De Rhodopsin-baserte prober kan ikke. Den tredje klasse av sensoren er like sterk som den første klasse, men krever tilsetning av en kjemisk quencher som er vanskelig å administrere in vivo. Vi vil derfor demonstrere oppkjøpet av en sonde med en enkelt FP (Bongwoori) 8 og en sonde som består av en FRET par (Nabi 2) 12. Gnage konstruerer i denne rapporten er sommerfugl versjoner av VSFP-CR (spenningssensitive fluorescerende proteiner - Clover-mRuby2) 11 som består av en grønn fluorescerende donor, Clover, og en rød fluorescerende akseptor, mRuby2, oppkalt Nabi 2,242 og Nabi 2,244 <sopp&gt; 12. Innledningen til disse typer opptakene skal gi forskerne en bedre forståelse av hvilken type informasjon GEVIs kan gi. <img alt="Figur 1" src="/files/ftp_upload/53566/53566fig1.jpg" /> Figur 1. To typer genetisk kodet spenningsindikatorer (GEVIs) avbildet i denne rapporten (A) En mono FP basert Gevi ha en trans-membran spenningsføler domene og et fluorescerende protein. (B) En FRET basert Gevi består av en trans-membran spenningsføler domene, en FRET donor og akseptor. <a href="https://www.jove.com/files/ftp_upload/53566/53566fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</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.

Forbigående transfekterte celler kan oppvise betydelige variasjoner i fluorescens intensitet og graden av plasmamembranen uttrykk. Selv på samme dekk noen celler vil ha varierende grad av intern fluorescens. Dette er mest sannsynlig på grunn av mengden av transfeksjon middel absorbert av cellen. Av og til fører til for mye uttrykk cellen til å oppleve den utbrettede protein respons som resulterer i apoptose 27 (lys, avrundet celler med høy indre fluorescens). Experimente...

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 membranpotensialet med to typer genetisk kodet Fluorescent Spenning Sensorer

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

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

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

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

Kvantitativ analyse av Synaptic vesicle Pool Etterfylling i Cultured lillehjernen Granule Neurons bruker FM Fargestoffer

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

Imaging neuronal Responses i Slice Produkter av vomeronasal Organ uttrykker en Genetisk Encoded Kalsium Sensor

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

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

Membranpotensialet Dye Imaging av ventromedial Hypothalamus nerveceller fra voksne mus for å studere Glukose 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 Intracellulær Ca<sup&gt; 2+</sup&gt; Signaler i striatale Astrocytter fra voksne mus Bruke genetisk kodet Kalsium Indikatorer

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

Optisk Kontroll av en neuronal Protein Ved hjelp av en genetisk kodet Unaturlig Amino Acid i nevroner

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

Avbildning av mitokondrie Ca2+ opptak i astrocytter og nevroner ved hjelp av genetisk kodede Ca2+ indikatorer (GECIer)

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