Based on screening in cultured neurons (Fig. 1), we chose three ultrasensitive GCaMP6 sensors (GCaMP6s, 6m, 6f; for slow, medium and fast kinetics, respectively) for characterization in vivo. These sensors vary in kinetics, with the more sensitive sensors having slower kinetics. Compared to GCaMP5G, the GCaMP6 sensors have similar baseline brightness and a 1.1-1.6 fold increase in dynamic range (ΔF/F0 at 160 action potentials). For small numbers of action potentials the most sensitive sensor, GCaMP6s, produced 7-fold larger signals (>10-fold larger than GCaMP3, Fig. 1b-e; Supplementary Table 1). Underlying this sensitivity gain are multiple factors (Supplementary Table 2). Compared to GCaMP5G, GCaMP6s exhibited 3-fold higher apparent affinity for calcium and 1.3-fold higher saturated fluorescence, with similar baseline fluorescence. Calcium-saturated GCaMP6s is 27% brighter than enhanced GFP (EGFP), its parent fluorescent protein. The fastest sensor, GCaMP6f, had 2-fold faster rise time and 1.7-fold faster decay time than GCaMP5G (Fig. 1f, g) (Supplementary Table 1). GCaMP6f is the fastest genetically-encoded calcium indicator for cytoplasmic free calcium in neurons, with sensitivity comparable to OGB1-AM (Fig. 1d-g). GCaMP6 indicators were more sensitive and/or faster
