Figure 5
From: Physiological responses of Daphnia pulex to acid stress
Calibration of cSNARF-1. Fluorescence emission spectra of a bicarbonate-buffered cSNARF-1 solution were acquired at various pH values (A). Fluorescence excitation was at 475 nm. Reiterative least-squares spectral resolution [137] (Additional file 4) was applied to the data to extract the spectra of the acid/base forms (B) and the pK'a value of cSNARF-1. The underlying model that relates the fraction of acid α to pH (D) is given by α = {H+}/(K'a + {H+}). The optimum pK'A value of 7.624 was reiteratively obtained by using the Nelder-Mead simplex algorithm [144]. The inclusion of an additive offset factor (E) and multiplicative scaling factor (F) into the optimization corrected for variations in CCD dark current, excitation light intensity, sample shape, and fluorophore concentration. The residuals (C) represent 'unexplained' spectral information (noise).