Initial Fluorescence

In TRIC experiments, the initial fluorescence denotes the fluorescence of a sample that is measured before the IR-Laser is switched on, i.e. at ambient temperature without additional sample heating. 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 of the parameters to judge the quality of an assay. In a dilution series, initial fluorescence is expected to be homogenous for all prepared dilutions.

The initial fluorescence values observed can be:

  1. Homogenous, within ± 20% of the average
  2. Have a random distribution with variations more than ± 20% (see Random Changes in Initial Fluorescence)
  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 case of homogenous initial fluorescence distributions no further measures are necessary.
  2. In case of Random Changes in Initial Fluorescence: Random changes often arise from sample inhomogeneities (such as Aggregation, air bubbles or poor sample mixing, for both see Liquid Handling), centrifugation and addition of Detergents are the most common remedial measures.
  3. and 4. In case of ligand concentration dependent increase or decrease of the initial fluorescence within the limit of ± 20% of the average, the fluorescence change can be ignored. In case the initial fluorescence change exceeds ± 20% of the average, per form a Specificity Test. Read the article on Ligand-Induced Fluorescence Change for more detailed information.