Confocal spot detection of presynaptic Ca²⁺ domains

Advances in rapid imaging of fluorescence probes are crucial to the study of neuronal signaling and information processing within the nervous system. Fluorescence indicators of cellular signaling (e.g., Ca²⁺ or voltage) can report changes on the millisecond timescale and signals can be localized to submicrometer structures. There are significant methodological challenges, however, involved in making measurements at such high temporal and spatial resolution. I describe here the principle and implementation of a confocal spot detection method for rapid and localized detection of fluorescence changes, which we have used for the detection of action potential-induced presynaptic Ca²⁺ domains.