Table 1.

Design and in vitro and in vivo properties of genetic probes of neural activity

Probe Design Ca sensor Working principle In vitro pKaaIn vitro Kd (μm) In vitro change (%) Change at 40 and 80 Hz (%) τ rise at 40 and 80 Hz (seconds)e τ decay at 40 and 80 Hz (seconds)e Visibility at restc
YC2.0 ECFP/EYFP CaM FRET 6.9 0.1 100 7.8 1.52 0.58/2.52 High
(SST) 11.0 11.6 (0.53) (0.47/3.39)
YC2.3 ECFP/citrine CaM FRET 5.7 0.1 100 5.5 0.72 0.53/3.48 High
4.3 9.4 (0.35) (0.53/2.37)
YC3.3 ECFP/citrine CaM FRET 5.7 1.5 100 5.9 1.07 0.62/3.02 High
9.6 (0.42) (0.51/2.84)
TN-L15 ECFP/citrine Tpn Cd FRET 5.7 1.2 140 6.9 0.49 1.29 High
8.2 (0.31) (1.29)
Camg1 Split EYFP CaM Ca-induced ∼7 7.0 700 (—) Very low
(SVQST) pKa change
Camg2 Split citrine CaM Ca-induced ∼7 5.3 700 (—) Moderate
pKa change
FP cpEYFP CaM Ca-induced ∼7 0.7 800 None
pKa change
IP cpEYFP CaM Ca-induced ∼7 0.2 down −6.7b 0.61 0.90 High
pKa change to 15 −8.9b (0.28) (0.98)
GCaMP 1.3 cpEGFP CaM Ca-induced ∼7 0.235 450 8.2 0.84 0.46 Moderate
pKa change 16.1 0.31 0.48
GCaMP 1.6 cpEGFP CaM Ca-induced ∼8.5 0.146 480 6.2 0.56 0.34 High
pKa change 17.8 0.16 0.35
SpH EGFP pKa ∼6 600 9.1 4.33 f Moderate
16.5 2.49 f

  • Differences in the molecular design of the probes, their spectral properties, Kd, the calcium binding moiety, and pH sensitivity are summarized. The in vitro performance, given in the original literature, is compared with the results from our in vivo experiments. In general, the signal changes observed in vivo tended to be much smaller than the maximum change observed in vitro. Subtraction of the background increases the calculated amplitudes only by a factor of 3-5, without increasing the SNR (see Fig. 4). Only small signals were, in particular, observed for Camg1 plus Camg2 and FP; the latter did not exhibit any detectable fluorescence. The other indicators exhibited reliable fluorescence changes relative to baseline. Their amplitude and time constant for the rise and decay are shown. Reliable time constants in the absence of saturation of the indicator are in bold. Besides these functional aspects, the fluorescence of the indicators at resting calcium (rightmost column) is an important parameter for the identification of expressing neurons in live experiments, in particular at the single-cell and subcellular level. SST, Mutations S65G, S72A, T203Y; SVQST, S65G, V68L, Q69K, S72A, T203Y; cp, circularly permutated.

  • ↵ a In dc probes, the relevant pKa of the longer wavelength chromophore.

  • ↵ b Calculated after subtraction of experiments without stimulus (see supplemental Fig. 2, available at www.jneurosci.org as supplemental material).

  • ↵ c Experimentally determined in presynaptic boutons ∼30 min after cutting the innervating axon.

  • ↵ d Tpn C from chicken skeletal muscle.

  • ↵ e Time constants were fit to a single exponential with R2 = 0.99. Only the decay of the cameleon indicators had to be fitted by a double-exponential function (τ1/τ2). The decay of TN-L15 signals followed a single-exponential time course.

  • ↵ f SpH did not return to baseline by the end of the experiment.