Confocal imaging of [Ca2+] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence

Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca2+ from cellular stores into the cytosol, a process that appears to be tightly controlled by changes in [Ca2+] within the store. A method to image free [Ca2+] within cellular organelles was devised, which provided the first quantitative confocal images of [Ca2+] inside the sarcoplasmic reticulum (SR) of skeletal muscle. The method exploits, for greater sensitivity, the dual spectral shifts that some fluorescent dyes undergo upon binding Ca2+. It was implemented with mag-indo-1 trapped in the intracellular organelles of frog skeletal muscle and validated showing that it largely monitors [Ca2+] in a caffeine-sensitive compartment with the structure of the SR cisternae. A tentative calibration in situ demonstrated an increase in the dye's dissociation constant, not unlike that observed for other dyes in cellular environments. This increase, together with other characteristics of the ratioing method, placed the half-signal [Ca2+] near 1 mm, a value suitable for cellular stores. Demonstrated advantages of the technique include accuracy (that of a calibrated ratiometric method), dynamic range and sensitivity (from the combination of two spectral shifts), spatial and temporal resolution, and compatibility with a vast array of visible dyes to monitor diverse aspects of cellular function. SEER (shifted excitation and emission ratioing) also provides a [Ca2+] -independent measure of dye concentration in the cell. Store and mitochondrial [Ca2+] ([Ca2+](SR) and [Ca2+](mito)) could be measured separately using the high spatial resolution of SEER. Evolution of [Ca2+](SR) was followed upon changes in cytosolic [Ca2+]([Ca2+](cyto)). At [Ca2+](cyto) = 100nm, [Ca2+](mito) remained near the lower limit of detection and [Ca 21+] SR stabilized at values that were submillimolar according to our tentative calibration. Steady [Ca2+](SR) was only slightly higher in 800 nm [Ca2+](cyto), and essentially did not decrease unless [Ca2+](cyto) was reduced below 10 nm. While the increase of [Ca2+](SR) was limited by loss through Ca2+ release channels, its decrease in low [Ca2+](cyto) was largely dependent on leaks through the SR Ca2+ pump.