Analysis of in vivo cSNARF-1 spectra. Example in vivo spectra (A) from a pH 7.8 acclimated Daphnia pulex exposed to ambient pH 7.8-3.0. The corresponding pH values were retrieved by a multicomponent analysis  (Additional file 4), which determines the composition of a mixture of components, given that the spectrum of each component is known. The component spectra (B) comprised the in vitro spectra of the acid/base forms of cSNARF-1 (measured in Daphnia hemolymph), a reference (autofluorescence) spectrum from non-injected animals, and an offset (background) spectrum. The multicomponent analysis yielded the fraction of the acid form (D), the relative chromophore (acid plus base) concentration (E), as well as the contributions of the reference and offset signals (F). The residuals (C) represent spectral information that could not be explained by the component signals.