Fluorescence ratio imaging of dynamic intracellular ionic signals

Abstract

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kdnear 200 nM) and selectivity for Ca2+to a versatile tetracarboxylate binding site3modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+indicators are crown ethers with cavity sizes chosen to select Na+over K+: Mg2+indicators use tricarboxylate binding sites truncated from those of the Ca2+chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.