Binding experiments of BF to TMRSA. Depicted is the amplitude of the cross-correlation function vs the BF to TMRSA concentration ratio. The concentration of TMRSA was in all experiments. The positive control (full circles) is shown with the best fitting model as a solid line (; ; , ; ; ; ). The negative control (open circles) is shown with the best fitting model as a dashed line (; , ; ; ; ). The shaded areas show the borders of the models which can fit the data with a change of of less than 50% of its minimum value. The model parameters have the following ranges: ; ; , ; ; ; . The two vertical gray lines delimit the [BF]/[TMRSA] concentration region in which the detection threshold for binding [Eqs. (22) and (23)].
Influence of the on the CCF. The amplitude of the CCF is shown vs the ligand to receptor concentration ratio. The curves were calculated for a standard fluorophore pair (fluorescence yields , , , ; binding stoichiometry 1:1; no quenching of ligand and receptor ).
Influence of impurities on the CCF. The amplitude of the CCF is shown vs the ligand to receptor concentration ratio. The curves were calculated for a standard fluorophores pair (fluorescence yields , , , ; binding stoichiometry 1:1; no quenching of ligand and receptor ) and for two different ’s (a, c, e: ; b, d, f: ). (a, b) fluorescent inactive impurities. (c, d) nonfluorescent active impurities. (e, f) Nonfluorescent inactive impurities. Curves for calculations assuming no impurities are given in solid lines. Curves for ligand impurities are given as dotted lines. Curves for receptor impurities are given as dashed lines.
Influence of cross-talk on the CCF. The amplitude of the CCF is shown vs the ligand to receptor concentration ratio. The curves were calculated for three different levels of cross-talk of the ligand fluorophores into the channel of the receptor fluorophores (fluorescence yield distributed over the two channels depending on cross-talk). The receptor fluorophores was assumed to have a cross-talk of 10% into the first channel (, ). The binding stoichiometry is 1:1 and no quenching of ligand and receptor were used, .
Sensitivity of SW-FCCS depending on increasing cross-talk of ligand fluorophores (dotted lines), receptor fluorophores (dashed lines), or both fluorophores simultaneously (solid lines). For these calculations a 1:1 binding stoichiometry and no quenching upon binding were assumed. For the ligand and receptor curves the cross-talk of one fluorophore was fixed at 10% while the cross-talk of the other flurophore was varied between 10% and 50%. At 50% cross-talk for a fluorophore the fluorescence intensity detected in the two detection channels is equal. For the ligand and receptor curves the cross-talk of both fluorophores was varied simultaneously between 10% and 50%. The fluorophores were assumed to result in 30 000 counts per second and particle over all detection channels. (a) The values of are depicted vs percentage of cross-talk. (b) The values of are depicted vs percentage of cross-talk. Maximum measureable ’s can be calculated from the data according to Eq. (22).
The influence of receptor labeling on the cross-correlation amplitudes. The graphs depict the cross-correlation amplitudes for a standard fluorophore pair (fluorescence yields , , , ; binding stoichiometry 1:1; no quenching of ligand and receptor, ). The ligand carries one fluorophores and the receptor can carry either 1–2 fluorophores (a, c) or 1–4 fluorophores (b, d). The ratios of receptors carrying 1 to fluorophores are given in the legends as . (a) and (b) depict the curves for a . (c) and (d) depict the curves for a .
Fluorescence intensities of the different particles in the detection channels 1 and 2 for standard solutions of . The average number of particles per observation volume in our setup for a solution is . From this number the values in brackets, the counts per particle and second, are calculated. The residual fluorescence after binding for the different particles is given by .
Maximum values with corresponding values, for a value of the detection threshold . Values are given for two fluorophore combinations: BF/QR and BF/TMRSA. With these values maximum and minimum detectable ’s can be calculated by Eqs. (34) and (35). All values were calculated using the spectroscopic data of Table I and using Eqs. (23), (32), and (33).
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