Fluo-2 AM Probes

Fura-2-acetoxymethyl ester

Fura-2-acetoxymethyl ester, often abbreviated Fura-2AM, is a membrane-permeant derivative of the ratiometric calcium indicator Fura-2 used in biochemistry to measure cellular calcium concentrations by fluorescence. When added to cells, Fura-2AM crosses cell membranes and once inside the cell, the acetoxymethyl groups are removed by cellular esterases. Removal of the acetoxymethyl esters regenerates "Fura-2", the pentacarboxylate calcium indicator. Measurement of Ca2+-induced fluorescence at both 340 nm and 380 nm allows for calculation of calcium concentrations based 340/380 ratios. The use of the ratio automatically cancels out certain variables such as local differences in fura-2 concentration or cell thickness that would otherwise lead to artifacts when attempting to image calcium concentrations in cells.

Fluo-2 AM Probes Figure 1. Fura-2-acetoxymethyl ester.

Fura-2 is a proportional fluorescent dye that binds to free intracellular calcium. It is the first dye widely used for calcium imaging and is still very popular. Fura-2 is excited at 340 nm and 380 nm, and the emission ratio at these wavelengths is directly related to the intracellular calcium content. Fura-2 emits light at 510 nm regardless of the presence or absence of calcium. The use of this ratio automatically eliminates confounding variables such as variable dye concentration and cell thickness, which makes Fura-2 one of the most appreciated tools for quantifying calcium levels. The high photon yield of furan 2 allowed the first real-time (video rate) measurement of calcium in living cells in 1986. Recently, genes encoding calcium indicators based on spectral variation of green fluorescent protein, such as Cameleons, supplemented the use of Fura-2 and other small molecule Probes for calcium imaging, but Fura-2 still maintains a faster speed.

Fluo-2 AM Probes Figure 2. Structure of Fura-2 Probes.

Indo-1

Indo-1 is a popular calcium indicator similar to Fura-2. Compared to Fura-2, Indo-1 has a double emission peak. The main emission peak in the calcium-free solution was 475 nm, while the emission shifted to 400 nm in the presence of calcium. It is widely used in flow cytometry. Pentapotassium salts are commercially available and are preferred over free acids due to their higher solubility in water. Although Indo-1 is not cell permeable, the pentaacetoxymethyl ester Indo-1 AM enters the cell and is cleaved to Indo-1 by endolactase. In 1985, Roger Y Tsien proposed the synthesis and properties of Indo-1.

Fluo-2 AM Probes Figure 3. Indo-1.

References:

Latt, SA.; et al. Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence. Journal of Histochemistry and Cytochemistry. 1975, 23 (7): 493–505.
Cheng, H.; et al. Calcium Sparks - Elementary Events Underlying Excitation-Contraction Coupling in Heart-Muscle. Science. 1993, 262 (5134): 740–744.
Grynkiewicz G.; et al. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 1985, 260 (6): 3440–50.