Fluorescent proteins are now regularly used to monitor a variety of aspects of biological systems. Some uses of fluorescent proteins involve, but are not limited to, monitoring the expression of certain genes or tracking cells during the course of an experiment. The main advantage that the use of fluorescent proteins has over other approaches is that the fluorescence can be monitored in real-time without destroying the sample, thereby allowing to monitor the location where fluorescence can be observed, taking several measurements to increase measurement accuracy, or to monitor aspects of an experiment over time. One indicator of the paradigm shift that resulted from the introduction of fluorescent proteins is that the 2008 Nobel Prize in chemistry was awarded for the discovery and development of the green fluorescent protein (GFP) .
The mechanism by which fluorescent proteins are used in experiments is that the DNA that encodes information for the fluorescent protein is embedded close to the DNA of genes whose expression one wants to monitor. The result of this is that fluorescent proteins are produced via transcription/translation whenever the targeted DNA is transcribed/translated. While it is not possible to directly measure the proteins of interest without disturbing the experiment, the fluorescence exhibited by the fluorescent protein can easily be measured using fluorescent microscopy or a plate reader. The measured fluorescence can then be correlated with the protein expression that one wants to monitor. As measuring fluorescence intensity is a non-destructive process, it is possible to construct a time profile of the fluorescence intensity and draw conclusions about the protein expression dynamics.
The green fluorescent protein was the first fluorescent protein to be isolated and used, however, there is now a variety of fluorescent proteins that can be commercially purchased (e.g., reference  lists 25 different fluorescent proteins). The emissions spectra of these different fluorescent proteins range from the far red, over red, orange, yellow, green, to cyan. While each of these fluorescent proteins can be used separately, the main advantage of having fluorescent proteins with different emissions spectra available is that it is possible to simultaneously monitor different aspects of the same experiment. However, the emissions spectra of the different fluorescent proteins overlap and any measurement involves contributions from all the fluorescent proteins used [3, 4]. This overlap in the emissions spectra is a trivial issue if only two different fluorescent proteins are used, i.e., one chooses two fluorescent proteins with minimal overlap in their spectra . However, extracting the contribution of individual types of fluorescent proteins from measurements becomes significantly more challenging as the number of different fluorescent proteins involved in experiments increases. One result of this is that current experiments are limited to a small number, usually two or at most three, of events that can be simultaneously monitored [6, 7].
This work tries to addresses the issues mentioned above and develops a technique for designing experiments involving several different types of fluorescent proteins. This is achieved by first formulating an optimization problem that determines the contribution of individual fluorescent proteins to the spectrum measured by a fluorescence plate reader. In a second step this optimization problem is extended to also perform experimental design, such that the accuracy of the predicted levels of the different fluorescent proteins is maximized. This experimental design involves the decision of which different fluorescent proteins should be used in an experiment.
In order to evaluate the procedure, data of the fluorescence spectra for the most commonly used fluorescent proteins have been collected. The procedure has been tested using simulations as well as experimental data created from mixtures of different types of E. coli reporters where each type of E. coli expressed a different fluorescent protein. Special attention has been given to the noise levels inherent in the experiments and measurements.
 Tsien, R.Y. The green fluorescent protein. Annual Reviews Biochemistry, Vol. 67, pp. 509–544 (1998).
 Lansford, R.; Bearman, G.; Fraser, S.E. Resolution of Multiple Green Fluorescent Protein Color Variants and Dyes Using Two-Photon Microscopy and Imaging Spectroscopy." Journal of Biomedical Optics, Vol. 6, No. 3, pp. 311-318 (2001).
 Dickinson, M.E.; Bearman, G.; Tille, S.; Lansford, R.; Fraser, S.E. Multi-Spectral Imaging and Linear Unmixing Add a Whole New Dimension to Laser Scanning Fluorescence Microscopy. BioTechniques, Vol. 31, No. 6, pp. 1272-1278 (2001).
 Shaner, N.C.; Steinbach, P.A.; Tsien, R.Y. A Guide to Choosing Fluorescent Proteins. Nature Methods, Vol. 2, No. 12, pp. 905-909 (2005)
 Haseloff, J. GFP Variants for Multispectral Imaging of Living Cells. Methods in Cell Biology, Vol. 58, pp. 139-151 (1998).
 Hu, C.-D. and Kerppola, T.K. Simultaneous Visualization of Multiple Protein Interactions in Living Cells Using Multicolor Fluorescence Complementation Analysis. Nature Biotechnology, Vol. 21, pp. 539-544 (2003).