Ratiometric Calcium Indicators

BTC

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Among the ratiometric calcium indicators, Fura-2 and Indo-1 are most commonly used. BTC is another excitation-ratioable calcium indicator. However, BTC can only be used for high calcium level detection due to its low affinity to calcium. In recent years, BTC has been increasingly used for monitoring potassium channels since BTC demonstrated an excellent fluorescence enhancement response upon binding thallium ion that selectively passes through potassium channels.

 

Fura-2

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Fura-2 is a ratiometric fluorescent dye which binds free intracellular calcium. It was the first widely-used dye for calcium imaging, and remains very popular. Fura-2 is excited at 340 nm and 380 nm, and the ratio of the emissions at those wavelengths is directly correlated to the amount of intracellular calcium. Regardless of the presence of calcium, Fura-2 emits at 510 nm. The use of the ratio automatically cancels out confounding variables, such as variable dye concentration and cell numbers, making Fura-2 one of the most appreciated tools to quantify calcium levels. Fura-2 is preferred for ratio-imaging microscopy, in which it is more practical to change excitation wavelengths than emission wavelengths.

 

Fura-8™

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Although Fura-2 has been widely used as the preferred excitation-ratioable calcium indicator, it has certain limitations, e.g., lower sensitivity compared to the single wavelength calcium dyes, such as Fluo-8^® and Cal-520™. AAT Bioquest has recently developed Fura-8™ to improve the calcium response of Fura-2. As demonstrated in Figures 18 and 19, Fura-8™ AM is more sensitive to calcium than Fura-2 AM. In addition, Fura-8™ has its emission shifted to longer wavelength (Em = 525 nm). Fura-8™ might be also used for the flow cytometric analysis of calcium in cells due to its excellent excitation at 405 nm that perfectly matches the violet laser line.

Key Features of Fura-8™:

• Fura-8™ responses to calcium the same way as Fura-2 does
• Red-shifted dual excitation wavelengths (354 nm/415 nm)
• Better excited at 405 nm for flow cytometric applications
• Compatible with common filter sets
• Higher signal/background ratio than that of Fura-2

Indo-1

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In contrast to Fura-2, Fura-8™ and BTC, Indo-1 is the preferred emission-ratioable dye for flow cytometry, where it is more practical to use a single laser for excitation (usually the 351"364 nm spectral lines of the argon-ion laser). The emission maximum of Indo-1 shifts from ~475 nm in Ca²⁺-free medium to ~400 nm when the dye is saturated with Ca²⁺ (see Figure 20). While Indo-1 is not cell permeant, its pentaacetoxymethyl ester, Indo-1 AM, enters the cell where it is cleaved by intracellular esterases to give Indo-1.