Fluorescence lifetime imaging of calcium flux in neurons in response to pulsed infrared light

Abstract

Pulsed infrared light can excite action potentials in neurons; yet, the fundamental mechanism underlying this phenomenon is unknown. Previous work has observed a rise in intracellular calcium concentration following infrared exposure, but the source of the calcium and mechanism of release is unknown. Here, we used fluorescence lifetime imaging of Oregon Green BAPTA-1 to study intracellular calcium dynamics in primary rat hippocampal neurons in response to infrared light exposure. The fluorescence lifetime of Oregon Green BAPTA-1 is longer when bound to calcium, and allows robust measurement of intracellular free calcium concentrations. First, a fluorescence lifetime calcium calibration curve for Oregon Green BAPTA-1 was determined in solutions. The normalized amplitude of the short and long lifetimes was calibrated to calcium concentration. Then, neurons were incubated in Oregon Green BAPTA-1 and exposed to pulses of infrared light (0-1 J/cm2; 0-5 ms; 1869 nm). Fluorescence lifetime images were acquired prior to, during, and after the infrared exposure. Fluorescence lifetime images, 64x64 pixels, were acquired at 12 or 24 ms for frame rates of 83 and 42 Hz, respectively. Accurate α1 approximations were achieved in images with low photon counts by computing an α1 index value from the relative probability of the observed decay events. Results show infrared light exposure increases intracellular calcium in neurons. Altogether, this study demonstrates accurate fluorescence lifetime component analysis from low-photon count data for improved imaging speed.