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).

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
<ol>
	<li>Imaging setup
	<ol>
		<li>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 imagi...

<ol>
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Membrane Biol. 19, 1-36 (1974).</li><li>Tasaki, I., Warashina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye-membrane+interaction+and+its+changes+during+nerve+excitation.">Dye-membrane interaction and its changes during nerve excitation.</a> Photochem Photobiol. 24, 191-207 (1976).</li><li>Grinvald, A., Hildesheim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSDI:+a+new+era+in+functional+imaging+of+cortical+dynamics.">VSDI: a new era in functional imaging of cortical dynamics.</a> Nat Rev Neurosci. 5, 874-885 (2004).</li><li>Jin, L., Han, Z., Platisa, J., Wooltorton, J. R., Cohen, L. B., Pieribone, V. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+FRET+dynamic+range+with+bright+green+and+red+fluorescent+proteins.">Improving FRET dynamic range with bright green and red fluorescent proteins.</a> Nat Methods. 9, 1005-1012 (2012).</li><li>Sung, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+fast+fluorescent+protein+voltage+sensors+by+optimizing+FRET+interactions.">Developing fast fluorescent protein voltage sensors by optimizing FRET interactions.</a> PLoS One. 10, e0141585 (2015).</li><li>Kralj, J. M., Douglass, A. D., Hochbaum, D. R., Maclaurin, D., Cohen, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+action+potentials+in+mammalian+neurons+using+a+microbial+rhodopsin.">Optical recording of action potentials in mammalian neurons using a microbial rhodopsin.</a> Nat Methods. 9, 90-95 (2012).</li><li>Flytzanis, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaerhodopsin+variants+with+enhanced+voltage-sensitive+fluorescence+in+mammalian+and+Caenorhabditis+elegans+neurons.">Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons.</a> Nat Commun. 5, 4894 (2014).</li><li>Hochbaum, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-optical+electrophysiology+in+mammalian+neurons+using+engineered+microbial+rhodopsins.">All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.</a> Nat Methods. 11, 825-833 (2014).</li><li>Gong, Y., Wagner, M. J., Zhong Li, J., Schnitzer, M. 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E., Mody, I., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+approach+to+measuring+electrical+activity+in+genetically+specified+neurons.">A hybrid approach to measuring electrical activity in genetically specified neurons.</a> Nat Neurosci. 8, 1619-1626 (2005).</li><li>Wang, D., McMahon, S., Zhang, Z., Jackson, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+voltage+sensor+imaging+of+electrical+activity+from+neurons+in+hippocampal+slices+from+transgenic+mice.">Hybrid voltage sensor imaging of electrical activity from neurons in hippocampal slices from transgenic mice.</a> J Neurophysiol. 108, 3147-3160 (2012).</li><li>Weigel, S., Flisikowska, T., Schnieke, A., Luksch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+voltage+sensor+imaging+of+eGFP-F+expressing+neurons+in+chicken+midbrain+slices.">Hybrid voltage sensor imaging of eGFP-F expressing neurons in chicken midbrain slices.</a> J Neurosci Methods. 233, 28-33 (2014).</li><li>Waters, J. C., Sluder, G., Wolf, D. 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The authors have nothing to disclose.

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...

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 cell types are difficult to stain. Genetically encoded voltage indicators (GEVIs) overcome these limitations by having the cells to be studied specifically express the fluorescent voltage-sensitive probe.
There are three classes of GEVIs. The first class of GEVI uses the voltage-sensing domain from the voltage-sensing phosphatase with either a single fluorescent protein (FP) 5-9 or a F&#246;rster resonance energy transfer (FRET) pair 10-12. The second class of sensors uses microbial rhodopsin as a fluorescent indicator directly 13-15 or via electrochromic FRET 16,17. The third class utilizes two components, the genetic component being a membrane anchored FP and a second component being a membrane bound quenching dye 18-20. While the second and third classes are useful for in vitro and slice experiments19,20, only the first class of sensors are currently useful for in vivo analyses 6.
In this report we will demonstrate the imaging of membrane potential using the first class of GEVIs (Figure 1) in vitro. This first class of voltage sensors is the easiest to transition to in vivo imaging. Since GEVIs utilizing a voltage-sensing domain fused to a FP are about 50-fold brighter than the rhodopsin class of sensors, they can be imaged using arc lamp illumination rather than requiring an extremely powerful laser. Another consequence of the disparity in brightness is that the first class of GEVIs can easily exceed the auto-fluorescence of the brain. The rhodopsin-based probes cannot. The third class of sensor is just as bright as the first class but requires the addition of a chemical quencher which is difficult to administer in vivo.
We will, therefore, demonstrate the acquisition of a probe with a single FP (Bongwoori) 8 and a probe consisting of a FRET pair (Nabi 2) 12. The FRET constructs in this report are butterfly versions of VSFP-CR (voltage-sensitive fluorescent proteins - Clover-mRuby2) 11 consisting of a green fluorescent donor, Clover, and a red fluorescent acceptor, mRuby2, named Nabi 2.242 and Nabi 2.244 12. The introduction to these types of recordings should give researchers a better understanding of the type of information GEVIs can provide.
<img alt="Figure 1" src="/files/ftp_upload/53566/53566fig1.jpg" /> 
Figure 1.&#160;Two Types of Genetically Encoded Voltage Indicators (GEVIs) Imaged in This Report (A) A mono FP based GEVI having a trans-membrane voltage-sensing domain and a fluorescent protein. (B) A FRET based GEVI comprised of a trans-membrane voltage-sensing domain, a FRET donor and acceptor. <a href="https://www.jove.com/files/ftp_upload/53566/53566fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</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>
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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.

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...

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

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 ...

The overall goal of this procedure is to optically report the membrane potential changes with a genetically encoded voltage indicator. This method will describe the strengths and weaknesses of imaging with different genetically encoded fluorescent probes of voltage. For instance, FRET-based probes will offer the advantage of ratiometric imaging but will give a lower signal.The main advantage of this technique is that we can visualize the neural activity in real time. Generally, individuals new to this method will struggle because the signal-to-noise ratio is not very large and there are several source of noise and multiple steps that can go wrong. New users are often confused and interference is taken as the real signals.Visual demonstration of this method is critical to ascertain the validity of the probes being used and what it actually indicates. For the setup, an image splitter is installed between the slow and fast CCD cameras for the imaging of FRET-based GEVI. Before the experiment, insert a filter cube with a dichroic mirror and two emission filters in the image splitter.Remove this second filter cube when imaging a GEVI with a single fluorescent protein. To begin the experiment, locate a healthy HEK293 cell that shows strong, localized membrane fluorescence compared to the internal fluorescence and avoid patching circular cells as they are either dividing or dying. Then apply a test pulse to check the current response.Afterward, lower the patch clamp pipette to right above the cell's surface. Then, slowly lower the pipette until it gently touches the cell membrane. The membrane resistance should increase to one to two megaohm.After that establish a gigaohm seal by gently applying negative pressure through the pipette. Once a gigaohm seal is achieved, set the pipette potential at a desired holding potential. Next, focus the high-speed CCD camera on the cell body.For the FRET-based GEVI recording, adjust the size of the split images to result in an evenly spaced view for each emission wavelength using the knobs on the image splitter. Then rupture the cell membrane by applying negative pressure in order to form the whole cell configuration. Now, open the imaging software.Then click acquire SciMeasure camera menu to open the CCD acquire page. Next, create a new data file to save the recording. Click analog output and then read an ASCII to open a pulse protocol file in order to conduct the imaging.Next click on average the internal repetitions to average the number of trials. Then close the analogue output page. Set the specific acquisition parameters on the CCD acquire page.After that input the specific values for number of frames for acquisition and number of trials. Then, click the take data optics plus BNC button to start the recording. While the voltage imaging is taking place monitor the oscilloscope to ensure stable whole-cell configuration throughout the recording.To calculate the fractional fluorescence change click file and then read data file to open a data file that was recorded in the previous step. The cell image in its resting light intensity should be seen on the right side. Next, click on show BNC's to show the current end voltage values.Change the page mode from RLI frame to frame subtraction in order to utilize the frame subtraction function to identify the pixels with responsive optical signals. Next, select two time points for subtraction and identify the cell area that shows signals in response to the membrane potential change. Next, designate the pixels that need to be analyzed by dragging or clicking each pixel.The graphical representation of the average fluorescence intensity from the selected pixels should appear on the left side of the software window. Divide the subtracted pixels with resting light intensity by clicking divide by RLI's button to acquire the fractional fluorescence change values. To export the data, remove the current end voltage graphs by unclicking show BNC's menu.Go to output, save traces as displayed ASCII to export the fluorescence trace in an ASCII file format for the curve fitting analysis. In this procedure, draw the fluorescence change versus voltage graph by plotting the fractional fluorescence changes in response to voltage in a data analysis program. After that fit the curve to a Boltzmann function in order to determine the voltage range of the optical signal by clicking analysis, fitting, sigmoidal fit then open dialog.Replot the normalized fractional fluorescence changes in response to voltage by using the data analysis program. To calculate the speed of the optical response, open the ASCII file and plot the fractional fluorescence change trace versus time. Then click on data selector in the data analysis software and select one time point corresponding to the beginning of a stepped voltage pulse and a second time point where the optical signal has reached the steady state.Fit this range to both a single and double exponential decay function by clicking analysis, fitting, exponential fit then open dialog and report the better fit. This figure shows the fluorescence change of an HEK cell expressing a single FP based GEVI Bongwoori. This is a typical fluorescence change in response to the stepped voltage pulses.Here an HEK293 cell was imaged with a high-speed CCD camera. This image shows the resting light intensity of a cell expressing Bongwoori. And this is a frame subtraction image indicating the pixels where fluorescence change was observed.Shown here is the optical recording of the induced action potentials from the mouse hippocampal primary neurons expressing Bongwoori. The action potentials were evoked under the whole-cell currents clamp mode. The fractional fluorescence change trace was selected from the pixels correlated to the soma.In this figure, the fluorescence change of an HEK cell expressing FRET-based GEVI shows the responses to the stepped voltage pulses in two wavelengths. Recorded at one kilohertz with a high-speed CCD camera. While attempting this procedure it's important to remember to test variable fluorescence levels since overexpression of fluorescence can affect the health of the cell.Following this procedure other methods like slicer codings can be performed in order to answer additional questions involving neuron activity of various circuits. After watching this video, you should have a good understanding of how to image membrane potential using different types of probe.

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10.6K Görüntüleme.  Seoul National University. The overall goal of this procedure is to optically report the membrane potential changes with a genetically encoded voltage indicator. This method will describe the strengths and weaknesses of imaging with different genetically encoded fluorescent probes of voltage. For instance, FRET-based probes will offer the advantage of ratiometric imaging but will give a lower signal.The main advantage of this technique is that we can visualize the neural activity in real time. Generally, individ...