In Spectral Shift experiments, the initial fluorescence of the sample is the fluorescence of the samples at the 650 nm channel. Ligand-induced changes in the fluorescence intensity at 670 nm have no effect on the ratiometric readout and, therefore, do not require a mathematical correction.

In TRIC experiments, on the contrary, the initial fluorescence is the fluorescence intensity at 670 nm measured before the IR-Laser is switched on, i.e. at ambient temperature without additional sample heating (F₀). The initial fluorescence is calculated from the non-normalized TRIC traces as an average over the data points obtained before IR-Laser activation.

The initial fluorescence is one parameter to judge the quality of an assay. In a dilution series, initial fluorescence is usually expected to be homogenous for all prepared dilutions.

The initial fluorescence can be:

1. Homogenous, within ± 20% of the average
2. Have a random distribution with variations of more than ± 20%
3. Decrease more than -20% with the ligand concentration
4. Increase more than +20% with the ligand concentration

Please follow these recommendations for the 4 scenarios mentioned above:

1.  In the case of homogenous initial fluorescence distributions, the assay is of excellent quality, and no further measures are necessary.
2. In case of random changes in the initial fluorescence: Since random changes often arise from sample inhomogeneities (such as Aggregation, air bubbles, or bad sample mixing), centrifugation and the addition of detergents are the most common measures.
3. and 4. In case ligand concentration-dependent increase or decrease of the initial fluorescence within the limit of ± 20% of the average, ignore the fluorescence change. If the initial fluorescence change exceeds ± 20% of the average, perform a Specificity Test.