What is Ratiometric Fluorescence?

Ratiometric fluorescence is the method where intensities at two or more wavelengths of an excitation or emission spectrum are measured to detect changes to local environment. Typically, a probe is used that is specifically sensitive to an environmental parameter such as ion concentration, pH, viscosity, or polarity.

The application of ratiometric dyes for finding probe-sensitive properties such as ion concentration can be used by measuring spectra or kinetics.

In the spectrum of Fura-2, you can see the fluorescence excitation has two peaks that directly reflect the binding of free ionic calcium in solution. At high free ionic calcium concentrations, the excitation peak at 340 nm is comparatively high. When free ionic calcium concentration is low, the excitation peak at 380 nm is going up in relation to the peak at 340 nm.

Taking the ratio of the intensities at two excitation wavelengths from these peaks directly correlates with the intracellular concentration of free ionic calcium. Measuring the ratio of the fluorescence at 510 nm when excited at 340 nm and 380 nm over time will give a time-based plot of how a solution is changing in calcium cation concentration. Cellular uptake of free ionic calcium is measured in this way.

There are other probes that have fluorescence spectra that vary in the same way with physical properties such as solvent polarity pH, and concentrations of other ions such as sodium, potassium, and magnesium.

Here is a graph of the ratio, indicating the intracellular calcium response of an atrium cell loaded with Fura-2. By taking the ratio between the two intensities over time, the concentration of calcium ions can be directly correlated to this kinetics curve.