Voltage indicator proteins and microscope for imaging rhodopsin fluorescence
System for optically measure cell membrane potential
Dr. Adam Cohen and colleagues have developed a novel protein-based indicator of cell membrane voltage with the potential to revolutionize the field of electrophysiology. Membrane potential plays a crucial role in biological processes such as nerve impulse transmission, mitochondrial and bacterial ATP production, and membrane transport. Currently, the most common way to measure the voltage across a cell membrane in real time is patch clamp, which involves placing electrical probes on both sides of the membrane. This technique is slow, painstaking, and limited to large cells without cell walls, and it often damages or kills the cells. An optical indicator of membrane potential is highly desirable, but current candidates are not sensitive enough, too slow, or toxic and difficult to install in the cell. Thus, the Cohen lab’s fast, sensitive, nontoxic, genetically encoded optical indicator represents a major breakthrough for electrophysiology.
Innovations and Advantages
1. Voltage Indicating Proteins (VIPs)
There are currently multiple tools to study electrical activity within the brain. Electrodes are commonly used to record activity from individual neurons. Small molecule dyes and proteins can be used to visually monitor calcium levels, which provide an indirect proxy for voltage across the cell membrane. Finally, dyes that are directly responsive to voltage changes have also been described. However, in each of these cases, there are significant limitations that include the inability to monitor a large population of cells simultaneously, poor response dynamics, lack of sensitivity, cellular toxicity, inadequate subcellular resolution, problems in cellular targeting, and incompatibility with long-term assessment. Having a tool that can overcome these limitations would represent a significant technical advance, and would undoubtedly speed discovery in neuroscience.
Adam Cohen and colleagues have now invented a novel method that addresses many of these limitations. This method makes use of Archaerhodopsin-3 (Arch), a specific member of an abundant class of naturally occurring proteins known as rhodopsins, which ordinarily convert light into electrical signals. Arch is normally found in a Dead Sea microorganism but when expressed in neurons, it is known to alter membrane potential upon exposure to light. Remarkably, Cohen et al discovered that Arch can be “run in reverse”: namely, that it can convert changes in membrane voltage into a detectable optical signal.
Importantly, when introduced genetically into neurons, Arch appeared non-toxic and produced a signal that is both fast and linear, enabling detection of action potentials in rat neurons in vitro even at the subcellular scale and in single trial recordings. However, when used in this way, Arch continued to produce small changes in membrane potential that could interfere with the normal electrical activity of the cell. Cohen et al therefore generated a mutant version of Arch, Arch(D95N), which no longer produced such changes and in fact produced an even stronger optical signal. Nevertheless, the optical signal of Arch and Arch(D95N) – which is emitted as fluorescent red light – is relatively dim when compared to other fluorescent proteins typically used for imaging cells in biology. Therefore, Cohen and colleagues custom built a powerful in-house microscope in order to be able to visualize electrical activity with Arch (See section 2).
The potential applications of this rhodopsin-based voltage indicator technology are far-reaching. The technology promises to be an exciting new tool to advance basic biological research in the fields of neurobiology as well as in other areas where cellular electrical activity is important in both normal and diseased states (e.g. cardiology). Perhaps less obviously but just as significantly, this technology also has great potential in the pharmaceutical industry to screen for: novel modulators of specific ion channels in neurons, cardiomyocytes and pancreatic beta cells; new compounds that direct embryonic stem cells down different trajectories; and toxicity of novel therapeutics (e.g. hERG compliance assays).
2. Microscope for Imaging Rhodopsin Fluorescence (MIRF)
Microbial rhodopsin proteins are a class of transmembrane proteins recently discovered to function as fluorescent indicators of membrane potential. Due to their unusual photophysical properties, a special-purpose microscopy system is required to make use of these proteins. Here we provide such a system. The key ingredients in the Microscope for Imaging Rhodopsin Fluorescence (MIRF) are:
1) An illumination system optimized to provide
a. High illumination intensity
b. Spatial, angular, temporal, and spectral control of the illumination
2) A detection system optimized to provide
a. High collection efficiency
b. Wide field of view with continuously variable magnification
c. Multi-spectral imaging
d. High sensitivity detection with frame rate > 1 kHz
3) A mechanical sample-positioning system optimized to provide
a. Accommodation for multi-well plates
b. Environmental controls (temperature and carbon dioxide)
c. Automatic focus, sample positioning, and sample exchange
d. Fluidic and microfluidic controls
4) Analysis algorithms to
a. Automatically identify cells on the basis of correlated fluctuations in membrane potential.
b. Estimate the spatiotemporal dynamics of membrane potential in one or more cells, using either single-wavelength or ratiometric imaging
c. Reconstruct the dynamics of an electrical impulse with resolution better than the frame-time of the camera using temporal superresolution.
5) Control software to allow a user to illuminate a particular collection of cells, single cell, or sub-cellular region with illumination of a user-selected wavelength.
6) Optionally a feedback system to adjust the illumination pattern in response to observed changes in fluorescence of the cells.
Kralj JM, Hochbaum DR, Douglass AD, Cohen AE. 2011. Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein. Science. 333(6040):345-8.
Kralj JM, Douglass AD, Hochbaum DR, Maclaurin D, Cohen AE. 2011. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat Methods. 9(1):90-5.
Cohen, Adam E.
Hochbaum, Daniel R.
Kralj, Joel M.
- Cardiovascular Diseases
- Drug Discovery Tools
- Imaging Agents
- Photonics, Optics and Optoelectronics
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Reference Harvard Case #4324