Name: imaging membrane potential with two types of genetically encoded fluorescent voltage sensors
Uploaded: 2016-02-04T14:00:00.000+00:00
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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).

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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 major key for imaging changes in voltage is the efficient expression of the GEVI in the plasma membrane. Intracellular expression creates a non-responsive fluorescence that reduces the SNR of the probe. Optimizing the transfection conditions vastly improves the consistency of the optical measurements. Indeed, when testing a novel GEVI it is advisable to also test a known probe to ensure that differences in optical activity are due entirely to the new probe and not the conditions of the cells.
Figure 6 shows the effect of internal fluorescence in decreasing the sensitivity of the voltage imaging, &#916;F/F value. Figure 6B shows &#916;F values an HEK cell expressing the single-FP based GEVI, Bongwoori. Trace 2 comes from region 2 (blue color) where relatively bright internal fluorescence is seen in Figure 6A. This trace has similar level of &#916;F value as trace 1. However, in Figure 6C where the traces in Figure 6B were divided by RLI values for &#916;F/F values, the trace 2 drops significantly due to the bright internal fluorescence which decreases &#916;F/F values. Fluorescence trace 3 has a very small &#916;F but also has a very small RLI value (F). The result is a misleading &#916;F/F signal that has a substantial increase in the noise of the recording. This example was included to demonstrate the &#916;F is also important. A large change in &#916;F/F is not helpful if F is very low to begin with.
The use of an image splitter enables the concomitant measurement of two wavelengths onto a single CCD camera. This greatly assists the measurement of FRET dependent fluorescent changes for probe development and reduces the cost of the setup. However, due to chromatic aberration, these two images will be slightly out of focus necessitating the need for an apochromatic objective. The more light the better SNR, so choosing objectives with the highest numerical aperture also improves fluorescence imaging. In addition, one can use stronger light sources such as light emitting diodes (LED) or laser to increase SNR. LEDs do not require a mechanical shutter.
In this report we show the optical signals from a single FP GEVI and a FRET-based GEVI. The main advantage of a FRET-based GEVI is that ratiometric imaging enables the reduction of correlated noise due to respiration and blood flow in vivo. The opposite polarity of the fluorescent signals confirms a real change in membrane potential. However, since the fluorescence intensities of the two chromophores often vary significantly, the SNR will also vary. This confounds the ratiometric analysis and limits its usefulness 28. There can also be a FRET-independent signal29. It is therefore sometimes better to use only the brighter fluorescent chromophore when using FRET to analyze changes in membrane potential.
Voltage imaging is more challenging than calcium imaging due to the speed and variability of the changes in membrane potential compared to calcium imaging which measures the calcium flux. The membrane potential changes in very fast time scales compared to the calcium ion flux, and subthreshold events in neurons cannot be measured with calcium imaging 30. Furthermore, the voltage probe must be in the plasma membrane to be effective which reduces the amount of probe available to optically report changes in the membrane potential. Consequently, the number of photons that can actually arrive at the CCD chip is relatively few. This requires the need for a high speed and low read-noise camera with high quantum efficiency and a probe that elicits a high SNR upon changes in membrane potential. By imaging cells in vitro, researchers will better understand the potential benefits and pitfalls of GEVIs before attempting in vivo measurements.

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.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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). The experimenter is therefore cautioned to test both bright cells and dim cells with as little internal fluorescence as possible since internal expression creates a non-responsive fluorescence that lowers the signal to noise ratio (SNR).
Another consideration is the maturation rate of chromophores. Figure 3 shows disparity in the fluorescence of the acceptor chromophore (mRuby2) which has a longer maturation time than the donor chromophore (Clover).
<img alt="Figure 3" src="/files/ftp_upload/53566/53566fig3.jpg" /> 
Figure 3. Confocal Images of HEK 293 Cells Transiently Transfected with a FRET Based GEVI, Nabi2.242. (A) Confocal images of an HEK cell showing membrane localized fluorescence from FRET donor and acceptor, (B) Confocal images showing a potential consequence of slow maturation of mRuby2. All four cells exhibit Clover fluorescence while only two exhibit mRuby2 fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The images were acquired by a confocal microscope. For the FRET donor, Clover, 488 nm laser was used for excitation and the emission was captured with 525/50 nm bandpass filter. The FRET acceptor, mRuby2, was illuminated with 561 nm laser for excitation and 595/50 nm bandpass filter was used for emission. The samples for confocal imaging were fixed with 4% paraformaldehyde/sucrose solution in phosphate buffered saline adjusted at pH 7.4 and then mounted with an anti-fade reagent.
Once the transfection conditions are optimized, the next source of variation comes from determining the region of interest to be analyzed. Using frame subtraction to identify the regions of the cell with the highest fluorescence change is often used to maximize the &#916;F/F value. An alternative is to select all of the pixels receiving light from the cell. This increases the number of pixels resulting in the reduction of noise, but decreases the signal size since non-responsive internal fluorescence is included. Both methods are fine as long as the experimenter remains consistent.
Figure 4A shows the fluorescence change of an HEK cell expressing a single-FP based GEVI, Bongwoori. This is a typical fluorescence change in response to stepped voltage pulses. From this data one can plot voltage sensitivity of the GEVI and determine the on and off &#964; constants at different voltages by fitting to a single or double exponential decay function. 16 trials are typically averaged when imaging HEK cells to improve the SNR of small voltage steps and to detect any potential bleaching during the recording. Those probes that give a robust signal at 100 mV can be tested in dissociated hippocampal neurons (Figure 4C). The fluorescence trace from the hippocampal neuron is a single trial.
<img alt="Figure 4" src="/files/ftp_upload/53566/53566fig4.jpg" /> 
Figure 4. Voltage Imaging with a Single FP Based GEVI Expressed in HEK 293 Cells and Hippocampal Primary Neurons. (A) &#916;F/F trace of a single FP based GEVI, Bongwoori, showing responses to stepped voltage pulses recorded at 1 kHz with a high speed CCD camera. (B) An HEK 293 cell imaged with the high speed CCD camera; (left) Resting Light Intensity (RLI) of a cell expressing Bongwoori &#38; (right) the frame subtraction image indicating the pixels where fluorescence change was observed. (C) Optical recording of induced action potentials from mouse hippocampal primary neurons expressing Bongwoori. The action potentials were evoked under whole cell current clamp mode. The &#916;F/F trace was selected from the pixels correlated to the soma. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A representative fluorescence readout of the donor and acceptor chromophores is shown in Figure 5A. The polarity of the fluorescence change is opposite for the donor and acceptor enabling ratiometric analysis to reduce correlated noise sources, e.g., movement. For example, movement correlated noise will exhibit a change in fluorescence in the same direction for both donor and acceptor fluorescence. While the FRET based probe is capable of ratiometric imaging, it is often better to only analyze the brighter chromophore. This is because the dimmer chromophore can substantially increase the noise of the recording. Figure 5B shows this effect. The frame subtraction in Figure 5B from the brighter Clover clearly shows where the optical signal is in the cell. In contrast, the frame subtraction of mRuby2 fluorescence shows higher degrees of noise throughout the cell. Therefore, only the donor signal of Clover is shown in a hippocampal neuron in Figure 5C.
<img alt="Figure 5" src="/files/ftp_upload/53566/53566fig5.jpg" /> 
Figure 5. Voltage imaging with a FRET based GEVI expressed in HEK 293 cell and hippocampal primary neurons.&#160;(A) &#916;F/F trace of a FRET based GEVI, Nabi2.242, showing responses at two wavelengths to stepped voltage pulses recorded at 1 kHz with a high speed CCD camera. (B) Top; the donor (Clover-RLI, Clover-frame subtraction) and acceptor (mRuby2-RLI, mRuby2-frame subtraction) images processed with two different thresholds for each FP, Bottom; the same threshold was used for both donor (Clover-RLI, Clover-frame subtraction) and acceptor (mRuby2-RLI, mRuby2-frame subtraction) images to illustrate the relative dimness of mRuby2. All the images were taken from the same HEK 293 cell expressing Nabi2.242. (C) The 520 nm wavelength trace of induced action potentials from a mouse hippocampal primary neuron expressing Nabi2.244 sensor. The &#916;F/F trace was selected from the pixels correlated to the soma. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53566/53566fig6.jpg" /> 
Figure 6. Consequences of Varying Light Levels for &#916;F and &#916;F/F Values.&#160;(A) An image of an HEK 293 cell expressing a single FP based GEVI, Bongwoori, shown in Resting Light Intensity (RLI), (B) the fluorescence traces showing kernel averaged &#916;F (Fx-F0) values from three different regions, region 1: membrane region with well localized fluorescence signal, region 2: a region with bright internal fluorescence, and region 3: region distant from optical signal, (C) the fluorescence traces showing kernel averaged &#916;F/F values from the same regions in (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Concepts</td>
			<td>Equations</td>
			<td>Remark</td>
		</tr>
		<tr>
			<td>Fractional fluorescence change (&#916;F/F)</td>
			<td><img alt="Equation 2" src="/files/ftp_upload/53566/53566eq2.jpg" /></td>
			<td>F1 = light intensity measured at a time point, F0 = light intensity measured at holding potential</td>
		</tr>
		<tr>
			<td>Boltzmann function</td>
			<td><img alt="Equation 3" src="/files/ftp_upload/53566/53566eq3.jpg" /></td>
			<td>y = &#916;F/F, V = membrane potential in mV, V1/2 is the membrane potential in mV at half-maximal&#160; &#916;F/F, A1 = the minimum value, A2 = the maximum value, dx = slope</td>
		</tr>
		<tr>
			<td>Double exponential decay function</td>
			<td>y = y0 + A1e -(t - t0)/ &#964;1 +&#160;A2e -(t - t0)/ &#964;2</td>
			<td>&#964;1, &#964;2 = time constants, A1,A2 = amplitudes, t, t0 = two time points, y0 = offset</td>
		</tr>
	</tbody>
</table>
Table 2. Equations

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.7K Views.  Seoul National University. A method for imaging changes in membrane potential using genetically encoded voltage indicators is described. 

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

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Developmental Biology

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS.

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

Biology

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Bioengineering

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Evaluating Electroporation Effects on Genetically Engineered Spiking HEK Cells

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could&#160;be efficiently evaluated.

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a ...

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring &#916;&#936;m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting &#916;&#936;m fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study &#916;&#936;m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

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

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