Figure 1

Fluorescence properties of genetically encoded pH-indicators. Spectra taken at different pHs are given for Pt-GFP (A), ratiometric pHluorin (C) and ecliptic pHluorin (E). Corresponding ratio curves are on the right hand side (B, D, F). A: Excitation spectra of Pt-GFP at λ_em = 540 nm. The dotted line represents the emission spectrum (λ_ex = 470 nm) corresponding to pH = 7.4. B: Dependency of fluorescence excitation ratios R(475ex/390ex; 540em) of Pt GFP on pH. C: Excitation spectra of ratiometric pHluorin at λ_em = 508 nm. The dotted line represents the emission spectrum (λ_ex = 390 nm) corresponding to pH 7.5. D: Dependency of fluorescence excitation ratios R(395ex/475ex; 508em) of ratiometric pHluorin on pH. E: Emission spectra of ecliptic pHluorin taken at different pH (λ_ex = 400 nm). The dotted line represents the excitation spectrum (λ_em = 508 nm) corresponding to pH 7.5. F: Dependency of fluorescence excitation ratios R(400ex; 511em/464em) of ecliptic pHluorin on pH. All spectra are normalized by their area. The grey curve in each set of spectra (A, C, E) represents the corresponding minimax spectrum (maximum fluorescence difference for each wavelength). Its minimum designates the isosbestic point. Ratio data (B, D, F) were fitted with a sigmoidal Boltzmann fit.