Imaging membranpotentialet med to typer af genetisk kodede Fluorescerende Spænding Sensorer
Summary
A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.
Abstract
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 Förster 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.
Introduction
Den største fokus i dette papir er at demonstrere den optiske billeddannelse af ændringer i membran potentialer in vitro ved hjælp af genetisk kodet fluorescerende proteiner. Imaging ændringer i membranpotentiale giver spændende mulighed for at studere aktiviteten af neuronale kredsløb. Når ændringer i membranpotentialet resultat i en fluorescensintensitet forandring, hver pixel i kameraet bliver et surrogat elektrode muliggør nonintrusive målinger af neuronal aktivitet. For over fyrre år har økologiske spændingsfølsomme farvestoffer været nyttigt til at observere ændringer i membranpotentialet 1-4. Men disse farvestoffer mangler cellulære specificitet. Desuden er nogle celletyper er vanskelige at farve. Genetisk indkodede Spændingsindikatorer (GEVIs) overvinde disse begrænsninger ved at have cellerne, der skal undersøges specifikt udtrykker det fluorescerende spændingsfølsomme probe.
Der er tre klasser af GEVIs. Den første klasse af GEVI bruger voltage-sensing domæne fra den spændingsdrevne sensing phosphatase med enten en enkelt fluorescerende protein (FP) 5-9- eller en Forster resonans (FRET) pair 10-12. Den anden klasse af sensorer anvender mikrobiel rhodopsin som en fluorescerende indikator direkte 13-15 eller via elektrokrome FRET 16,17. Den tredje klasse anvender to komponenter, den genetiske komponent er en membran forankret FP og en anden komponent er en membranbundet quenching farvestof 18-20. Mens den anden og tredje klasser er nyttige til in vitro og skive eksperimenter 19,20, kun den første klasse af sensorer er i øjeblikket anvendelige til in vivo analyser 6.
I denne rapport vil vi demonstrere billeddannelse af membranpotentialet ved hjælp af den første klasse af GEVIs (figur 1) in vitro. Denne første kategori af spændingssensorer er den nemmeste at overgangen til in vivo-afbildning. Da GEVIs utilizing en spænding-sensing domæne fusioneret til et FP er ca. 50 gange lysere end den rhodopsin klasse af sensorer, de kan afbildes ved hjælp af belysning buelampe snarere end at kræve en ekstremt kraftig laser. En anden konsekvens af forskellen i lysstyrke er, at den første klasse af GEVIs let kan overstige autofluorescens af hjernen. De rhodopsin-baserede prober ikke kan. Den tredje klasse af sensoren er lige så lyse som den første klasse, men kræver tilsætning af en kemisk quencher, som er vanskelige at administrere in vivo.
Vi vil derfor demonstrere erhvervelsen af en sonde med en enkelt FP (Bongwoori) 8 og en sonde, der består af et FRET-par (Nabi 2) 12. Den FRET konstruktioner i denne rapport, er sommerfugl versioner af VSFP-CR (spændingsfølsomme fluorescerende proteiner – Clover-mRuby2) 11, der består af et grønt fluorescerende donor, Kløver, og en rød fluorescerende acceptor, mRuby2, opkaldt Nabi 2,242 og Nabi 2,244 <sop> 12. I indledningen til disse typer af optagelser bør give forskerne en bedre forståelse af den type oplysninger GEVIs kan give.
Figur 1. To typer af genetisk kodede Spændingsindikatorer (GEVIs) Trykte i denne rapport (A) En mono FP baseret GEVI have en trans-membran spænding-sensing domæne og et fluorescerende protein. (B) En FRET baseret GEVI består af en trans-membran spænding-sensing domæne, et FRET donor og acceptor. Klik her for at se en større version af dette tal.
Protocol
Representative Results
Discussion
Nervesystemet anvender spænding på flere forskellige måder, inhibering forårsager en mindre hyperpolarisering, synaptisk input forårsager en mindre depolarisering og et aktionspotentiale resulterer i en relativt stor spændingsændring. Evnen til at måle ændringer i membranpotentiale af GEVIs giver lovende potentiale analysere flere komponenter af neuronale kredsløb samtidigt. I denne rapport demonstrerer vi en grundlæggende metode til billeddannelse ændringer i membranpotentialet hjælp GEVIs.
<p class="j…Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| Inverted Microscope | Olympus | IX71 | |
| 60X objective lens (numerical aperture = 1.35) | Olympus | UPLSAPO 60XO | |
| Excitation filter | Semrock | FF02-472/30 | For voltage imaging of super ecliptic pHluorin in Bongwoori |
| Dichroic mirror | Semrock | FF495-Di03-25×36 | |
| Emission filter | Semrock | FF01-497/LP | |
| 75W Xenon arc lamp | CAIRN | OptoSource Illuminator | LEDs and lasers are also effective light sources |
| Slow speed CCD camera | Hitachi | KP-D20BU | |
| Dual port camera adaptor | Olympus | U-DPCAD | |
| High speed CCD camera | RedShirtImaging, LLC | NeuroCCD-SM | |
| Image splitter | CAIRN | Optosplit 2 | |
| Excitation filter | Semrock | FF01-475/23-25 | For voltage imaging of FRET pair based GEVI consisting of Clover and mRuby2) |
| Dichroic mirror | Semrock | FF495-Di03-25×36 | |
| Emission filter | Chroma | ET520/40 | |
| Dichroic mirror | Semrock | FF560-FDi01-25X36 | |
| Emission filter | Chroma | ET645/75 | |
| Vibration isolation system | Kinetic systems | 250BM-IC, 5702E-3036-31 | |
| Patching chamber | Warner instruments | RC-26G, 64-0235 | |
| #0 Micro Coverglass (22x40mm) | Electron Microscopy Sciences | 72198-20 | |
| Temperature controller | Warner instruments | TC-344B | |
| #0 (0.08~0.13mm) – 10mm diameter glass coverslip | Ted Pella | 260366 | |
| Lipofection agent | Life Technologies | 11668-027 | |
| Calcium phosphate reagent | Clontech – Takara | 631312 | |
| Patch clamp amplifier | HEKA | EPC 10 USB amplifier | |
| Multi-channel data acquisition software | HEKA | Patchmaster | |
| Image acquisition and analysis software | RedShirtImaging | Neuroplex | |
| Spreadsheet application software | Microsoft | Microsoft Excel 2010 | |
| Data analysis software | OriginLab | OriginPro 8.6.0 | |
| Demagnifier | Qioptiq LINOS | Optem standard camera coupler 0.38x SC38 J clamp | |
| Confocal microscope | Nikon | Nikon A1R confocal microscope | |
| Anti-fade reagent | Life Technologies | P36930 |
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