Since being introduced in 1989,1 fluo-3 imaging has revealed the spatial dynamics of many elementary processes in Ca2+ signaling.2-4 Fluo-3 has also been extensively used for flow cytometry;5 for experiments involving photoactivation of “caged” chelators, second messengers, and neurotransmitters;6,7
and for cell-based pharmacological screening.8 The most important properties of fluo-3 in these applications are an absorption spectrum compatible with excitation at 488 nm by argon-ion lase r sources, and a very large fluorescence intensity increase in response to Ca2+ binding. Fluo-4 is an analog of fluo-3 with the two chlorine substituents replaced by fluorines. This fairly minor structural modification result s in increased fluorescence excitation at 488 nm and consequently higher signal levels for confocal microscopy, flow cytometry, and microplate screening applications. Fluo-5F, fluo-5N, and fluo-4FF are analogs of fluo-4 with lower Ca2+-binding affini ty, making them suitable for detecting intracellular calcium levels in the 1 μM–1 mM range that would saturate the response of fluo-3 and fluo-4. The Fluo-4 dextrans consist of fluo-4 coupled to a biologically inert dextran carrier (molecular weight = 10,000), providing a new and potentially valuable tool for measuring Ca2+ transients in presynaptic terminals arising from long axonal projections in heterogeneous fiber tracts.9 Molecular Probes offers two reactive forms of Fluo-4, an iodoacetamide and a cadaverine. The fluo-4 iodoacetamide can react with sulfhydryl groups to form unique fluorescent Ca2+-sensitive probes, including proteins, peptides, and thiol-modified surfaces. The aminecontaining Fluo-4 cadaverine can react with aldehydes, ketones, and activated esters.