PLATERO - Green Fluorescence Calibration in Plate Readers v1

One of the most common sources of information in Synthetic Biology is the data coming from plate reader fluorescence measurements. These experiments provide a measure of the light emitted by certain fluorescent molecules, such as the Green Fluorescent Protein (GFP). However, these measurements are generally expressed in arbitrary units, and are affected by the measurement device gain. This limits the range of measurements in a single experiment and hampers the comparison of results among experiments. In this work, we provide a calibration protocol to express fluorescence measures in concentration units of a reference fluorophore. The protocol removes the gain effect of the measurement device on the acquired data. In addition, the fluorescence intensity values are transformed into units of concentration using a Fluorescein calibration model. Both steps are expressed in a single mathematical expression which returns normalized, gain independent, and comparable data, even if the acquisition was done at different device gain levels. The protocol embeds a Lineararity and Bias Analysis that provides an assessment of the uncertainty of the model estimations, and a Reproducibility and Repeatability analysis that evaluatesthe sources of variability originating from the measurements and the equipment. All the functions used to build the model, exploit it with new data, andperform the uncertainty and variability assessment are available in an open access repository.