Increases and decreases in membrane potential play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, and cell signaling. Potentiometric probes are important tools for studying these processes and are generally characterized as slow- or fast-response probes.
Molecules that change their structure in response to the surrounding electric field can function as fast-response probes for the detection of transient (millisecond) potential changes. Slow-response dyes function by entering depolarized cells and binding to proteins or membranes.
Increased depolarization results in additional dye influx and an increase in fluorescence, while hyperpolarization is indicated by a decrease in fluorescence. Fast-response probes are commonly used to image electrical activity from intact heart tissues or measure membrane potential changes in response to pharmacological stimuli. Slow-responding probes are often used to explore mitochondrial function and cell viability.
Plasma Membrane Potential Indicators
Human HEK 293 cells were loaded with FluoVolt Membrane Potential Dye. Cells were imaged with 10 millisecond illumination pulses and images acquired with 2X binning. Traces show fluorogenic responses as cells are depolarized (A) at 2 second intervals from –100 mV to +30 mV or (B) in single steps from –80 mV to 0 mV at 2 second intervals.
Differentiated NG-108 cells were loaded with FluoVolt Membrane Potential Dye. Cells were imaged with 10 millisecond illumination pulses and images acquired with 2X binning. The three selected traces (B) show fluorogenic responses from the dye as selected cells (A) spontaneously depolarize and repolarize in culture.
FluoVolt Membrane Potential Dye
FluoVolt Membrane Potential Dye is a fast-response probe with a superior potential-dependent fluorescence response. The response is fast enough to detect transient (millisecond) potential changes in excitable cells, and the probe generates a signal change in excess of 25% per 100 mV. FluoVolt Membrane Potential Dye can be used for imaging electrical activity from intact heart tissues, mapping membrane potentials along neurons and muscle fibers, or measuring potential changes in response to pharmacological stimuli.
Traditional Fast-Response Probes
ANEP dyes fluoresce in response to electrical potential changes in their environment. These are fast-response probes that change their electronic structure, and consequently their fluorescence properties, in response to changes in the surrounding electric field. The response is fast enough to detect transient (millisecond) potential changes in excitable cells, including single neurons, cardiac cells, and intact brains. However, the magnitude of their potential-dependent fluorescence change is often small (2–10% fluorescence change per 100 mV).
Slow-Response Probes
The slow-response potential-sensitive probe, DiBAC4(3) enters depolarized cells where it binds to intracellular proteins or membrane and exhibits enhanced fluorescence and a red spectral shift. Increased depolarization results in additional influx of the anionic dye and an increase in fluorescence. Conversely, hyperpolarization is indicated by a decrease in fluorescence. This bis-oxonal has an excitation maximum of 490 nm and emission maximum of 516 nm. DiBAC dyes are excluded from mitochondria because of their overall negative charge, making them superior to carbocyanines for measuring plasma membrane potentials.
Mitochondrial Membrane Potential Indicators to detect apoptosis
A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. We offer a range of Molecular Probes products specifically designed to assay mitochondrial membrane potential.
Mitochondrial potential assays for flow cytometry
Mitochondrial potential assays for imaging
| FluoVolt Membrane Potential Dye | di-3-ANEPPDHQ | Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol (DiBAC4(3)) | Voltage Sensor Probes Set, DiSBAC2(3) and CC2-DMPE | |
|---|---|---|---|---|
| Response type | Fast-response | Fast-response | Slow-response | Slow-response |
| Readout | Fluorescence increase with depolarization | 2 excitation wavelengths; ratiometric readout | Fluorescence increase with depolarization | 2 emission wavelengths; ratiometric readout |
| Range | 25% per 100 mV | 2–10% per 100 mV | 1% per 1 mV | 1% per 1 mV |
| Response time | Sub-millisecond | Sub-millisecond | 20 ms | 500 ms |
| Standard filter set | FITC | FITC/Cy5 | FITC | FITC/Cy5 |
| Ex/Em (nm) | 522/535 | 465/635 | 490/516 | 490/516 |
| Bibliography | Citations | Citations | Citations | Citations |
| Platforms | Imaging | Imaging | Imaging, Microplate | Microplate |
| Usage notes | Bright signal with higher S/N enables faster sampling rates for sub-millisecond response | Lower signal intensity requires a slower sampling rates to achieve adequate S/N | DiBAC dyes are excluded from mitochondria, making them ideal for measuring plasma membrane potentials | FRET-based assay optimized for high throughput |
| Format | 1 kit | 1 mg | 25 mg | 1 mg |
| Cat. No. | D36801 | B438 | K1046 |
Resources
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