Fig. 1
From: Quantitative determination of fluorescence labeling implemented in cell cultures
Method workflow. Left, outside boxes: depiction of the steps needed in the experiments. In panels A and B, each of the four columns represent a different experiment; grey circles in cells represent unlabeled molecules, which initially are N('); magenta and green circles are molecules labeled with either of two different dyes (n(')1 or n(')2 in number, where subscript 1 or 2 indicates respectively the first or second labeling reaction). On the bottom, the two microscopy detection channels (in green and magenta) are schematically represented for a single field of view for each experiment to visualize the same cells labeled with both the two dyes at the end of the whole labeling procedure. Box A: method to be used when no labeling efficiency information is available; both experiments must be performed, inverting the order of labeling while keeping constant the reaction conditions cA and cB for the magenta and the green probe respectively. From the ratios of the labeled molecules r and r' in the two cases, it is possible to extract the labeling efficiencies eA(cA) and eB(cB) for the two probes in the used conditions (see main text and Eqs. (1) and (2)). Box B: method to measure eA in varying conditions (e.g., γA, γA') of the “magenta” dye, keeping constant the conditions cB for the “green” one used as control with known efficiency eB(cB). Molecules are labeled sequentially with the test and the control probe and the ratio between the number of molecules labeled with the two dyes is evaluated. Each experiment yields the labeling efficiency eA(γA), eA(γA') for the tested probe in the used condition (Eq. (3)). Created with BioRender.com