The design and properties of a series of calcium indicators which shift from rhodamine-like to fluorescein-like fluorescence on binding calcium

Abstract

The prototype for a new series of ratio-mode fluorescence indicators of cytosolic free Ca2+ concentration ( [Ca2+Ii) has been developed. The fluorophore, termed FluoRhod,S is a hybrid containing elements of the closely related fluorescein and rhodamine structures. The novel feature of the prototype indicator, FluoR hod-2, which incorporates a tetracarboxylate chelating element similar to that of 1,2bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid (BAPTA), is that it shifts from rhodamine-to fluorescein-like fluorescence on binding Ca2? The synthesis and properties of a series of tetracarboxylate derivatives of the FluoRhod fluorophore are described which led to the FluoRhod-2 structure with an apparent dissociation constant for Ca2+ and pK, adjusted to meet the requirements for a [Ca2+Ii indicator in the ratio mode. The excitation and emission maxima of FluoRhod-2 are 537 nm and 566 nm in the absence of Ca2+ and 480 nm and 537 nm in the presence of Ca2+. The indicator can be used in either the dual excitation or dual emission mode or the ratio of these two ratios can be used by making dual excitation and dual emission measurements. The brightness of FluoRhod-2 is comparable to that of fura-2 with the advantage of excitation in the visible range. FluoRhod-2 is insensitive to pH from 6.5 to 7.5 when used in the ratio mode, irrespective of the extent to which the indicator is complexed to Ca2?The uses and limitations of the current generation of fluorescent indicators of cytosolic free Ca2 + concentration ([Ca2 'Ii) are well established. Fura-2 has been most used; it is about 30 times brighter than the prototype indicator, quin2.' The calibration problem is overcome because the excitation spectrum of fura-2 shifts when Ca2+ is bound and therefore allows [Ca2+Ii to be measured directly from the ratio of fluorescent emission intensities at two excitation wavelengths (usually 340 and 380 nm).2 The limitations of fura-2 are mainly the short wavelengths of the excitation maxima and the insolubility of the acetoxymethyl ester (AME) derivative 3,4 used to achieve cell permeation. To overcome this the indicator is frequently dispersed in a surfactant, pluronic F-127.4*5 Fura-2 AME is hydrolysed more slowly than quin2 AME and under some conditions bright spots of particulate fluorescence are observed in Both the slow hydrolysis and the association of the indicator with intracellular structures may be due to the hydrophobicity of fura-2 AME. The most recent versions of fluorescent indicators for [Ca2 'Ii are based on the fluorescein or rhodamine chromophores incorporated into BAPTA derivatives to give fluo-3 and rhod-2 with excitation wavelengths at 499 nm and 521 nm respectively.8 Calcium binding causes an increase in fluorescence emission but as the unchelated indicators are not significantly fluorescent the indicators cannot be used for ratio measurements of [Ca2 'Ii. There are, however, major potential advantages of using the fluorescein or rhodamine fluorophores for [Ca2 +Ii indicators t Abbreoiations used: AME, ace...

A tautomeric zinc sensor for ratiometric fluorescence imaging: Application to nitric oxide-induced release of intracellular zinc

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Synthesis of isomerically pure carboxylate- and sulfonate-substituted xanthene fluorophores

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