FAQ

  • What is ratiometric voltage imaging?

    Ratiometric imaging uses 2 separate wavelengths of light (can be either excitation, emission light, or both). Fluorescence intensities of the 2 separate channels are simply divided (ratio) to produce the ratiometric image or movie.

  • What makes a dye ratiometric/are all dyes ratiometric or compatible with ratiometric imaging?

    Ratiometric voltage-sensitive dyes are electrochromic and shift their spectra in response to changes in voltage. See Stark-effect.

  • Can ratiometric voltage-sensitive dyes be used non-ratiometrically, using single excitation and emission wavelength bands?

    Yes, all ratiometric voltage-sensitive dyes can also be used non-ratiometrically. “Red-edge” excitation yields the best sensitivity and this wavelength is shown for each of the ElectroFluors on our website.

  • Which dyes are ratiometric?

    Most dyes from Potentiometric Probes are ratiometric including the ElectroFluors. Some calcium dyes such as Fura-2 are also ratiometric.

  • Which dyes are non-ratiometric?

    Voltage-sensitive dyes that use other voltage sensing mechanisms such as TMRM, FluoVolt, and Berst.

  • What are the advantages of ratiometric voltage imaging?

    The main advantages of ratiometric voltage imaging are:

    - Artifact (such as motion) reduction/elimination

    - Reliability

    - Quantitativeness

    - Signal-to-noise

  • What are the disadvantages of ratiometric voltage imaging?

    Ratiometric imaging can reduce the frame rate by a factor of 2, but workarounds are available. The imaging setup is more complicated but we offer a versatile kit that is adaptable to your needs.

  • How complicated is it to set up a ratiometric imaging system?

    Systems are straightforward to set up, requiring the appropriate selection of off the shelf components, (cameras, filters, etc.) and Potentiometric Probes can provide support or a complete system including hardware, software, and data analysis.

  • Does it matter which channel is divided by which channel?

    When taking the ratio of 2 signals, you have the choice of which signal is the denominator or on the bottom. For multiplexed, dual excitation wavelength imaging, it is best to put the “red-edge” signal on the bottom since it is inverted.

  • How severe are motion artifacts? Are they always a dealbreaker?

    Cardiac tissues and cell cultures contract and this movement can be a dealbreaker for imaging, but not always. Imaging large fields-of-view can reduce or practically eliminate the artifact. However, motion artifacts depend on the strength of contraction, and for peace of mind, reliability, and the other advantages mentioned above, we recommend ratiometric voltage imaging whenever possible.

  • How is signal-to-noise affected by ratiometric imaging?

    Signal-to-noise ratio is a comparison of signal and noise amplitudes. Ratiometry can affect both the signal and the noise, but with care, the result can be a ~30% improvement in signal-to-noise. This is, of course, only in the absence of motion artifact, because the presence of motion artifact can be considered a noise source that can overwhelm the signal.