FAQ

What algorithm do you use to scale the calcium-independent signal (violet illumination) to the calcium-dependent signal (blue illumination)?

Why do you use 410nm (calcium-independent) and 488nm (calcium-dependent) illumination?

The relevant references for using 405 nm and 490 nm illumination are from fiber photometry: Zalocusky et al. Nature 2016 (http://web.stanford.edu/group/dlab/media/papers/zalocuskyNature2016.pdf), Kim et al. Nature Methods 2015 (http://web.stanford.edu/group/dlab/media/papers/kimNMeth2016.pdf) and Lerner et al. Cell 2015 (http://web.stanford.edu/group/dlab/media/papers/lernerCell2015.pdf). The Lerner et al paper in Fig S4 characterizes GCaMP6f at 405 nm. It does not show in the Tian paper whether it is exactly 405 or 410 nm that is the isosbestic point, but in practice it does not seem to matter.
Being narrower around the isosbestic point is presumably better. We think the 410/488 nm strategy is better because the signals should be more comparable (being fluorescence) than between the oxy/deoxy isosbestic point, which is usually measuring reflection/absorption. The ratio between oxy/deoxy absorption is roughly the same at 410 and 488 nm, although the absolute magnitude of both is different.
In both cases you are trying to measure the _fluorescence_ signal, which is green fluorescence > 495 nm. In our experience, the hemodynamic signal appears to effectively acts as a filter on top of this fluorescence, making it dimmer or brighter. This change in fluorescence intensity should be comparable between the 410 and 488 nm channels, except that the 488 nm channel also has Ca2+ flluorescence on top of that.