What is the difference between Fura-2 and Fluo 4?

Differences Between Fura-2 and Fluo-4

Fura-2 and Fluo-4 are both fluorescent indicators used for measuring intracellular calcium (Ca²⁺) concentrations, but they have distinct properties and applications. Below are the key differences between them:

Chemical Structure and Mechanism

• Fura-2 is a ratiometric indicator, meaning it can be excited at two different wavelengths (340 nm and 380 nm), and the ratio of the fluorescence intensities at these wavelengths is used to calculate Ca²⁺ levels. This property allows for more accurate measurements that are less affected by indicator concentration, cell path length, or dye leakage.
• Fluo-4 is a single-wavelength indicator, excited at approximately 494 nm, and emits fluorescence at around 516 nm. Its fluorescence intensity increases with Ca²⁺ concentration, making it simpler to use but more susceptible to variations in dye concentration and photobleaching.

Sensitivity and Specificity

• Fura-2 has a high sensitivity to Ca²⁺ changes and can measure a wide range of Ca²⁺ concentrations. It is particularly useful for detecting subtle or rapid changes in intracellular Ca²⁺ levels.
• Fluo-4 is less sensitive than Fura-2 but offers a brighter fluorescence signal, making it suitable for detecting higher Ca²⁺ concentrations and for applications where a strong fluorescent signal is preferred.

Applications

• Fura-2 is widely used in research that requires precise quantification of Ca²⁺ dynamics over a broad range of concentrations, especially in studies involving fast or small changes in Ca²⁺ levels.
• Fluo-4 is preferred for imaging applications where high brightness and ease of use are important, such as in high-throughput screening or when using confocal microscopy to visualize Ca²⁺ waves or sparks.

Photobleaching

Both dyes are subject to photobleaching, but Fluo-4 tends to bleach more rapidly than Fura-2 under similar conditions. This can affect long-term imaging experiments or when using intense illumination.