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Seeing Cellular
Excitation

Whether for basic research or drug discovery, precise measurement of voltage changes at the cell membrane is essential for understanding function, pathology, and potential therapeutic effects in electrically active cells.

The Question
Seeing Cellular Excitation





Above: HEK cells with patch clamp. Field is 120 microns wide. Image courtesy of Christopher Werley.

Ideal techniques for studying cellular voltage changes provide millivolt sensitivity and millisecond response times, preferably at subcellular resolutions.8,9,10

The traditional "gold-standard" method of patch clamping provides single-cell data with outstanding sensitivity and time resolution. Though very precise, patch clamping is also labor intensive and slow, and not well suited to multi-cell and longitudinal studies.3,8,9,10,12

Bottom: Spontaneous electrical wave propagation through HEK cells expressing Nav 1.3 and KIR 2.1, as shown through voltage-sensitive dye imaging. Above panel, waves originated as self-reinforcing spirals. Image courtesy of Christopher Werley.

Advances in optical live-cell imaging promise advances from better understanding of neuronal networks to high-throughput screening of potential CNS and cardiac drugs.1,2,3,4 Voltage-sensitive fluorescent dyes and genetically encoded voltage indicator proteins provide bright, membrane-localized signal.11 Fast, sensitive scientific CMOS detectors can image those signals at millisecond time scales.

How Many Frames per Second?
Try this decision tree for the right frame rate for visualizing five types of time-sensitive experiments using the ORCA-Flash4.0 camera—read now.

Above: Still image of a spiral wave, which serves as a stable source for periodic electrical spiking.

Above: Spiral wave center. The fluorescence change is plotted in color on a dark red (low voltage) to yellow (high voltage) color map. The induced fluorescence change is overlaid on a grayscale image of the average fluorescence level. Field-of-view is 400x400 microns, playback slowed five-fold. Video courtesy of Christopher Werley.

However, capturing transient, millisecond events such as action potentials requires an indicator that responds very quickly to voltage changes. A new experimental method from Park, et al.6 of Adam Cohen's lab at Harvard University makes it possible to screen candidate indicator molecules for response times down to 4 ms, while also checking for the bright signal and efficient localization to the plasma membrane needed.

Based on a monoclonal strain of human embryonic kidney (HEK) cells that exhibit spontaneous action potentials, the new platform enables researchers to image electrophysiological events—from subcellular changes to coordinated firing across entire cellular networks—at video speeds.

Read the Paper
Park, J, et al. Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells. PLoS One 8(12), e85221 (2013).
Voltage-Sensitive Dyes and Genetically-encoded Voltage Indicators
THE BARRIERS
Seeing Cellular Excitation

Patch-clamp recording uses an electrode inside a micropipette to measure electrical potential across the cell membrane, in whole cells or even individual ion channels, thanks to a high-resistance seal between the pipette tip and cell membrane. Whole-cell methods used to monitor action potentials provide precise quantitation, beyond the limits of the current optical assay, but take much more time and manual effort.2,6,12

At the Speed of Life
Learn how Ahrends and colleagues visualize activity in the living zebrafish brain using the speed and resolution of the ORCA-Flash4.0 camera—read now.

Above: Typical patch-clamp setup allows detection of current across the cell membrane.

Above: iPS cardiomyocyte membrane voltage dynamics. Video from Hamamatsu.

Optical methods allow high-throughput observation of activity from the subcellular level to multicellular networks. However, they have been limited by the challenge of developing voltage-sensitive indicators that are free from toxicity and pharmacologic effects, localized to the plasma membrane specifically, and highly efficient in producing a bright signal in response to voltage changes—within milliseconds.3,6

The Right Camera for the Job
The ORCA-Flash4.0 scientific CMOS camera's wide field of view, fast frame rates and high effective quantum efficiency enabled live cell video of action potentials using fluorescence. But what if luminescent markers are needed? Compare sCMOS and EM-CCD technology to find the right camera for the job—read more.
THE SOLUTION
Seeing Cellular Excitation

Above: Plane wave propagation. A video of voltage recorded using the voltage sensitive dye VF2.1.Cl showing a plane wave, the most common electrical propagation pattern. Brightness is proportional to voltage-induced change in fluorescence. The field of view is 3x6 mm and the playback is slowed five-fold from real time. Image courtesy of Christopher Werley.

Park, et al., demonstrated the sensitivity and responsiveness of the new assay by confirming its results against published attributes for several well-characterized dyes and indicator proteins, as well as patch-clamp studies of their own. Then they tested the assay in screening for candidate genetically expressed protein indicators, using mutant strains of bacterial rhodopsin, called Archeorhodopsin. With excellent sensitivity and speed, Archeorhodopsin has been used to resolve individual action potentials in cultured neurons.5 Park and colleagues identified several new variants with even better speed and sensitivity.

Video microscopy of single cells at 60x showed a wavefront of action potentials crossing the HEK cell monolayer. The assay showed good temporal resolution, even at single-cell resolution. The depolarization wave took about 1 ms to cross a cell, with the voltage rise taking about 3 ms. The mean action potential rise time was 2.5 ms +/- 1.3 ms.

To get the apparent response time of each candidate indicator, the researchers convolved camera exposure time (~1 ms), cellular voltage rise time (~2.5 ms) and true response time of known indicator. Based on that, the authors estimate that the new method can measure response times down to and below about 4 ms. That's faster than most transgenic voltage indicators in the literature so far, making this assay an excellent tool to filter candidates for the fastest fluorescent voltage indicators.

Left: Validating the assay with voltage-sensitive fluorescent dyes and genetically encoded fluorescent voltage indicators.

Experimental Conditions
THE POSSIBILITIES
Seeing the Living Brain
“… intracellular recordings of the full electrophysiological repertoire … are, at present, obtained only by sharp or patch microelectrodes. These, however, are limited to single cells at a time and for short durations.”
Spira, et al.8
“Voltage imaging, on the other hand, could in principle capture the entire picture: reading out the electrical activity of each neuron in the circuit, including sub-threshold excitatory and inhibitory events, for all different cell types.”
Peterka, et al.3
“Patch-clamp measurements in a fluorescence microscope provide quantitative and precise data, but manual patch clamp is laborious and slow.”
Peterka, et al.6
“… these probes allow us to measure and control neuronal signals with spatial resolution and genetic specificity that already greatly surpass those of electrophysiology.”
Park, et al.6
“Optical recording with a voltage-sensitive dye is advantageous where membrane potential must be recorded in many sites at once.”
Zecevic, et al.11

Park and colleagues’ new platform can quantify fluorescent marker response times of between about 4 and 50 ms, and provides both high speed and high sensitivity visualization of electrophysiological events. Validated on fluorescent voltage-sensitive dyes and genetically expressed indicators, this assay

could be used to test any type of fluorescent voltage indicator. The HEK cell model could also prove useful for basic research into cellular electrical activity and drug discovery, observing candidate drug effects on the spiking waveform and propagation.

References

01. Scanziani, M and Häusser, M. Electrophysiology in the age of light. Nature 461(7266), 930–939 (2009).

02. Dunlop, J. et al. Ion channel screening. Comb. Chem. High Throughput Screen. 11(7), 514-22 (2008).

03. Peterka, D.S, et al. Imaging Voltage in Neurons. Neuron 69(1), 9–21 (2011).

04. Kim, S.A. and Jun, S.B. In-vivo Optical Measurement of Neural Activity in the Brain. Exp. Neurobiol. 22(3): 158–166 (2013).

05. Kralj, J. et al. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nature Methods 9, 90–95 (2012).

06. Park, J. et al. Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells. PLoS One 8(12), e85221 (2013).

07. Pucihar, G and Kotnik, T. Measuring the induced membrane voltage with di-8-ANEPPS. J. Vis. Exp. 33: e1659 (2009).

08. Spira, M.E. and Hai, A. Multi-electrode array technologies for neuroscience and cardiology. Nat. Nanotechnol. 8(2), 83-94 (2013).

09. Szentandrássy, N. et al. Powerful technique to test selectivity of agents acting on cardiac ion channels: the action potential voltage-clamp. Curr. Med. Chem. 18(24), 3737-56 (2011).

10. Nauen, DW. Methods of measuring activity at individual synapses: a review of techniques and the findings they have made possible. J. Neurosci. Methods. 194(2), 195-205 (2011).

11. Zecevic, D, et al. Imaging nervous system activity with voltage-sensitive dyes. Curr. Protoc. Neurosci. Chapter 6:Unit 6.17 (2003).

12. Karmazínová, M and Lacinová, L. Measurement of cellular excitability by whole cell patch clamp technique. Physiol Res. 59 Suppl 1:S1-7 (2010).

13. Bébarová M. Advances in patch clamp technique: towards higher quality and quantity. Gen Physiol Biophys. 31(2):131-40 (2012).