Fig 1. Parameters necessary for calculation of ratio factor A for Cerulean and Venus fluorescent proteins.

(A) Spectra of the optical components in the excitation path of the imaging system. S_src(λ), S_ex(λ), S_dichr(λ), S_obj(λ) are spectra for the light source (Polychrome V monochromator, TILL Photonics, yellow), excitation filter (Chroma 69008x, blue), dichroic mirror (Chroma 69008bs, green) and objective (Olympus UAPO 40XOI3/340, black), respectively, mounted on Olympus IX71 microscope. Point-by-point multiplication of spectra for each optical element yields an optical function of microscope excitation light path shown by a violet trace. This excitation optical function of the particular imaging system can be used for calculation of a ratio factor A for a wide range of different fluorescent labels. (B) Further point-by-point multiplication of the optical function (violet), normalized Cerulean absorption spectrum $\left(S_{abs}^C(\lambda \right)$ (blue), and a spectrum of monochromator slit chosen for a given experiment (orange) gives a function of excitation path for Cerulean (black). Integration of this function and multiplication by the extinction coefficient for Cerulean absorbance results in $E_{ex}^C$, necessary for estimation of the ratio factor A (see Eq 1). (C) Spectra of the optical components in the emission path of the imaging system. S_obj(λ),S_dichr(λ),S_em(λ),S_det(λ) are objective (Olympus UAPO 40XOI3/340, black), dichroic mirror (Chroma 69008bs, green), and emission filter (Chroma 69008m, dotted red) transmittance and detector (QImaging ExiBlue, brown) sensitivity, respectively. Point-by-point multiplication of spectra for each optical element gives an optical function of microscope emission path shown by a red bold trace. This emission optical function of the particular imaging system can be used for calculation of a ratio factor A for a wide range of different fluorescent labels. (D) Further point-by-point multiplication of the optical function (red dashed trace; left Y axes) and Cerulean emission spectrum, integral of which is normalized to 1, $\left(S_{em}^C\right(\lambda \left)\right)$ (blue dashed trace; right Y axes) gives the function of emission path for Cerulean (black trace, right Y axes). Integration of this function and multiplication by Cerulean quantum yield results in $E_{em}^C$.