Whereas Spectral Shift measures the fluorescence ratio of two channels (650 nm and 670 nm), TRIC uses only one channel (670 nm) to measure the changes in the chemical environment of the fluorophores to determine molecular interactions qualitatively and quantitatively. They both measure a ratio: Spectral Shift measures the isothermal changes in the fluorescence of two channels (650 nm and 670 nm), whereas TRIC measures the fluorescence before and after an IR-Laser induced temperature changes.

Sometimes the initial fluorescence signal (the Fluorescence at 670 nm for Spectral Shift, and the fluorescence measured before the IR-Laser induced temperature change for TRIC) can also be ligand-dependent. There are two major reasons for an increase in initial fluorescence of the labeled target molecule when a ligand is added:

• The interaction between ligand and target molecule changes the chemical environment of the fluorophore in a way that it shows a higher fluorescence intensity.
• The ligand itself is fluorescent in the red spectral range measured by Dianthus instruments.

Empirical investigation has revealed that initial fluorescence changes induced by an added ligand that are below a threshold of ± 20 % do not significantly affect the obtained signals. In single dose measurements changes below this threshold are therefore ignored. Should the measured initial fluorescence of the target in presence of a ligand exceed +20 % the ligand is flagged as being auto fluorescent. To distinguish between ligand autofluorescence and a binding-induced effect, additional control experiments are required. In affinity measurements, initial fluorescence changes that influence the measured signals are compensated for in the Kd Fit Model. It is still advisable to determine the origin of initial fluorescence changes in a control experiment.

The simplest control experiment is to measure a solution of the ligand molecule in the absence of target. If fluorescence is observed the ligand is indeed auto fluorescent. If no fluorescence is observed for the ligand alone, the increase in target fluorescence is likely binding related. One option to work around an auto-fluorescent ligand is to reverse the setup, using the ligand as fluorescent target molecule and titrating the molecule that was initially the target if a non-fluorescent variant of it is available.