Development and in vitro characterization of ratiometric and intensity-based fluorescent ion sensors.

Fluorescent ion sensors are quite valuable in experimental biology. The development of new sensor molecules requires determination of spectral properties (absorption bands, fluorescence excitation, and emission maxima) in order to characterize the type of optical response to the target ion. This optical response type and magnitude are used, in combination with solutions of buffered ion of precisely manipulated concentration, to determine the in vitro affinity for the target ion. Buffered aqueous ion solutions of appropriate pH and ionic strength are necessary to predict the performance of new sensors in biological applications. A series of novel benzoxazole-BAPTA calcium sensors, in addition to Rhod-3 (a new version of rhod-2), are described and their optical responses to calcium ion characterized.

Fluorescent metal ion indicators based on benzoannelated crown systems: a green fluorescent indicator for intracellular sodium ions.

The synthesis and metal binding properties of cation-sensitive fluorescent indicators intended for biological applications are described. The increase of the crown ether ring size enhances the affinity for larger cations, but weakens the fluorescent response and selectivity. A compound having a 15-crown-5 chelator directly attached to a 2,7-difluoroxanthenone fluorophore loads into live cells and responds to sodium ion concentration changes with large fluorescence increases in the visible wavelength range.

Novel fluo-4 analogs for fluorescent calcium measurements.

We report new fluorescent calcium indicators based on fluo-4. Attachment of a carboxamide or methylenecarboxamide moiety to the BAPTA chelator portion of fluo-4 allowed for the attachment of dextrans, protein-reactive moieties, and biotin. In particular, a high affinity fluo-4 dextran conjugate was prepared and shown to be functional in brain slices. All new probes were characterized spectroscopically and exhibited large fluorescence increases upon calcium-binding. The biotinylated version of fluo-4 formed a persistent streptavidin complex which still responded to increasing calcium concentrations with a large fluorescence increase.

Fluorescent sodium ion indicators based on the 1,7-diaza-15-crown-5 system.

A series of novel sodium ion-sensitive fluorescent reagents suitable for biological applications is described. The chelator nitrogen atom substituents affect the selectivity and affinity of cation binding, while the nature of the fluorophore determines the type of fluorescent response to metal ion chelation.

Presynaptic calcium measurements at physiological temperatures using a new class of dextran-conjugated indicators.

Presynaptic calcium (Ca(pre)) has been studied extensively because of its role in triggering and modulating neurotransmitter release. Although calcium regulation and calcium-driven processes can be strongly temperature dependent, technical difficulties have limited most studies of Ca(pre) to temperatures well below the physiological range. Here we assessed the use of membrane-permeant acetoxymethyl (AM) indicators and dextran-conjugated indicators for measuring Ca(pre) at physiological temperatures. A comparison of these two types of indicators loaded into parallel fibers of rat cerebellar slices revealed striking differences. AM indicators were rapidly extruded from axons and presynaptic terminals and therefore cannot be used for long-term measurements at high temperatures. In contrast, dextran-conjugated indicators were retained within parallel fibers and are therefore well suited to measuring Ca(pre) at physiological temperatures. The limited number of dextran indicators available prompted us to synthesize three new indicators that show peak emission in the red (575-600 nm). These indicators allow for simultaneous use of multiple calcium indicators that can be readily distinguished on the basis of excitation and emission wavelengths, use of excitation and emission wavelengths that are relatively insensitive to tissue autofluorescence, and measurements in systems with expression of green fluorescent protein (GFP). Thus we find that dextran-conjugated indicators are well suited to long-term recordings of Ca(pre) at physiological temperatures and that the development of new red indicators greatly extends their utility.

Detection of nitric oxide from pig trachea by a fluorescence method.

A nitronyl nitroxide radical covalently linked to an organic fluorophore, pyrene, was used to detect nitric oxide (NO) from freshly excited tissues. This approach is based on the phenomenon of the intramolecular fluorescence quenching of the fluorophore fragment by the nitroxide. The pyrene-nitronyl (PN) reacts with NO to yield a pyrene-imino nitroxide radical (PI) and NO(2). Conversion of PN to PI is accompanied by changes in the electron paramagnetic resonance (EPR) spectrum from a five-line pattern (two equivalent N nuclei) into a seven-line pattern (two nonequivalent N nuclei). The transformation of the EPR signal is accompanied by an increase in the fluorescence intensity since the imino nitroxide radical is a weaker quencher than the nitronyl one. The results indicate that the fluorescence measurements enable detection of nanomolar concentrations of NO compared to a sensitivity threshold of only several micromolar for the EPR technique. The method was applied to the determination of NO and S-nitroso compounds in tissue from pig trachea epithelia. The measured basal flux of S-nitroso compounds obtained from the tissues was about 1.2 nmol/g x min, and NO-synthase stimulated by extracellular adenosine 5'-triphosphate produced NO flux of 0.9 nmol/g x min.