Time-Resolved Imaging Reveals Heterogeneous Landscapes of Nanomolar Ca2+ in Neurons and Astroglia

Maintaining low intracellular calcium is essential to the functioning of brain cells, yet the phenomenology and mechanisms involved remain an enigma. We have advanced a two-photon excitation time-resolved imaging technique, which exploits high sensitivity of the OGB-1 fluorescence lifetime to nanomolar Ca2+ concentration ([Ca2+]) and enables a high data acquisition rate in situ. The [Ca2+] readout is not affected by dye concentration, light scattering, photobleaching, micro-viscosity, temperature, or the main known concomitants of cellular activity. In quiescent tissue, standard whole-cell configuration has little effect on resting [Ca2+] inside neuronal dendrites or inside astroglia dye-filled via gap junctions. Mapping basal [Ca2+] in neurons and astrocytes with submicron resolution unveils heterogeneous concentration landscapes that depend on age and preceding activity. The rich information content represented by such landscapes in acute slices and in vivo promises to unveil the hitherto unexplored, potentially fundamental aspects of brain cell physiology.