Fluorescence dye preparation

Rose Bengal (Sigma 330000) and Rhodamine B (Sigma 252425) were prepared in ethanol at 100 μM concentrations. The two fluorescence dyes have similar emission spectra while they have very different fluorescence lifetime and quantum yields. Four mixtures of RhdmB and RB were prepared and measured with different volumetric proportions. RhdmB intrinsically has a higher quantum yield (0.7) compared to RB (0.11) therefore the 1:1, 1:5, 1:10, and 1:50 volumetric proportions for RhdmB:RB have been chosen.

To evaluate the ability of TRFS to differentiate the endogenous fluorescence mixtures mimicking biological tissues, three biomolecules including β-Nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH) (Sigma N8129), flavin adenine dinucleotide disodium salt dehydrate (FAD) (Sigma F6625), and Protoporphyrin IX (PpIX) (Sigma P8293) were used. The NADH, FAD, and PpIX were dissolved in Dimethyl sulfoxide (DMSO) (Sigma D4540) solutions individually, while PpIX was dissolved only in DMSO. The concentration of NADH, FAD, and PpIX are 1 mM, 0.1 mM, and 0.1 mM, respectively. All samples were measured after they are freshly prepared at room temperature. Four combinations of fluorophore mixtures including NADH[1]:FAD[1], NADH[1]:FAD[5], NADH[1]:FAD[10], NADH[1]:FAD[1]:PpIX[1] all in DMSO were measured and analyzed.