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

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

The major focus of this paper is to demonstrate the optical imaging of changes in membrane potentials in vitro using genetically encoded fluorescent proteins. Imaging changes in membrane potential offers the exciting possibility of studying the activity of neuronal circuits. When changes in membrane potential result in a fluorescence intensity change, each pixel of the camera becomes a surrogate electrode enabling nonintrusive measurements of neuronal activity. For over forty years, organic voltage-sensitive dyes have been useful for observing the changes in membrane potential ^1-4. However, these dyes lack cellular specificity. In addition, some ce....

Protocol

Ethics statement: The animal experiment protocol was approved by the Institutional Animal Care and Use Committee at KIST animal protocol 2014-001.

1. Equipment Setup

1. Imaging setup
   1. Place an inverted fluorescence microscope on a vibration isolation table. Use a high magnification (60X oil immersion lens with 1.35 numerical aperture) objective lens and a filter cube equipped with a dichroic mirror and filters suitable for the fluorescent proteins used for the voltage imaging.
      Note: This setup uses an inverted microscope in order to employ objectives with higher NA, but upright microscopes can also be used. Indeed,....

Results

Transiently transfected cells can exhibit significant variation in fluorescence intensity and the degree of plasma membrane expression. Even on the same coverslip some cells will have varying levels of internal fluorescence. This is most likely due to the amount of transfection agent absorbed by the cell. Occasionally, too much expression causes the cell to experience the unfolded protein response resulting in apoptosis ²⁷ (bright, rounded cells, with high internal fluorescence.......

Discussion

The nervous system uses voltage in several different ways, inhibition causes a slight hyperpolarization, synaptic input causes a slight depolarization and an action potential results in a relatively large voltage change. The ability to measure changes in membrane potential by GEVIs offers the promising potential of analyzing several components of neuronal circuits simultaneously. In this report we demonstrate a fundamental method for imaging changes in the membrane potential using GEVIs.

A maj.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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-Di03-25x36
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-Di03-25x36
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 (22 x 40 mm)	Electron Microscopy Sciences	72198-20
Temperature controller	Warner instruments	TC-344B
#0 (0.08 ~ 0.13 mm) - 10 mm 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