How does Fura-2 detect calcium?

Understanding Fura-2 for Calcium Detection

Fura-2 is a fluorescent dye widely used in biological and biochemical research for detecting and quantifying intracellular calcium (Ca2+) levels. Its mechanism of action is based on its ability to bind to calcium ions, which results in a change in its fluorescence properties. This change can be quantitatively measured, allowing researchers to monitor calcium dynamics within cells.

How Fura-2 Works

Fura-2 is a ratiometric dye, meaning it can absorb light at two different wavelengths, and the ratio of the emitted fluorescence at these wavelengths changes in response to calcium binding. Specifically, Fura-2 has two excitation peaks, one at around 340 nm and another at 380 nm, while its emission peak is around 510 nm.

When Fura-2 is free in the cytoplasm, it preferentially absorbs light at 380 nm. Upon binding to calcium ions, its chemical structure changes, leading to a preference for absorbing light at 340 nm. By measuring the fluorescence intensity ratio of light emitted after excitation at these two wavelengths, researchers can determine the concentration of free calcium ions within the cell.

Procedure for Detecting Calcium with Fura-2

  • Cells are loaded with Fura-2 by incubating them with the acetoxymethyl (AM) ester form of the dye, which is cell-permeable.
  • Once inside the cell, esterases cleave the AM groups, trapping the dye within the cytoplasm.
  • The cells are then illuminated alternately at 340 nm and 380 nm wavelengths.
  • The emitted fluorescence at 510 nm is measured for both excitation wavelengths.
  • The ratio of these fluorescence intensities is calculated, which is directly proportional to the intracellular calcium concentration.

This ratiometric measurement compensates for potential issues such as dye concentration, cell thickness, and photobleaching, making Fura-2 a reliable tool for studying calcium signaling in cells.

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