Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity Calcium Ruby and its dextran conjugate

The limited choice and poor performance of red-emitting calcium (Ca2+) indicators have hampered microfluorometric measurements of the intracellular free Ca2+ concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca2+ indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH2 linker arm. The low-affinity variant (KD,Ca ∼30 μM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca2+ transients. While Calcium Ruby is a mitochondrial Ca2+probe, its conjugation, via the NH2 tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca2+ probe with an affinity matched to microdomain Ca2+ signals. As an example, we show depolarization-evoked Ca2+ signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca2+ measurements.