Short Communication Experimental design of systems involving multiple fluorescent protein reporters Loveleena Bansal a,1 , Randall Nelson a , Eric Yang a , Arul Jayaraman a , Juergen Hahn b,c,n a Department of Chemical Engineering, Texas A&M University, College Station, TX 77843, United States b Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, United States c Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, United States HIGHLIGHTS  Developed mathematical guidelines to select fluorescent reporters for simultaneous use.  Mixtures of reporters are made using fluorescent protein-expressing E. coli strains.  Contribution of reporters to overall mixture intensity is calculated using linear unmixing.  D-optimal design is used to select proteins to maximize the estimation accuracy. article info Article history: Received 10 January 2013 Received in revised form 25 April 2013 Accepted 7 June 2013 Available online 14 June 2013 Keywords: Systems engineering Optimal design Fluorescent proteins Parameter identification Biological and biomolecular engineering Mathematical modeling abstract Fluorescent proteins have found widespread applications for analysis of biological systems as they can be used to track various events within living cells. Multiple fluorescent proteins are also simultaneously used to monitor different aspects of biological systems. However, extensive overlap in the emission spectra of the fluorescent proteins poses challenges in extracting the contribution of individual proteins to overall fluorescence intensity measurements. This work addresses this issue by deriving a computa- tional formulation for extracting the contribution of fluorescence intensities of individual reporters to the overall measurements taken using a plate reader. Then, this formulation is used for deriving an experimental design criterion for choosing sets of fluorescent proteins such that the accuracy of the estimated contribution of different fluorescent proteins is maximized. The results are validated using two sets of experimental data involving different sets of fluorescent proteins. This work represents the first quantitative study that evaluates experimental design for selection of fluorescent proteins to use simultaneously for multiple-labeling applications. & 2013 Elsevier Ltd. All rights reserved. 1. Introduction 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 genes, protein localization and protein–protein interactions (Lippincott-Schwartz and Patterson, 2003; Van Roessel and Brand, 2002). 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 monitoring of the location where fluorescence can be observed, taking several measurements to increase measurement accuracy, or to monitor aspects of an experiment over time. There are now a number of fluorescent proteins that are commercially available (Nowotschin et al., 2009; Shaner et al., 2005) which can be simultaneously used for profiling of biological systems. The main advantage of concurrently using multiple fluorescent reporters, with different emissions spectra, is that it is possible to simultaneously monitor different components of a system or their interactions. For instance, a number of researchers have used multiple fluorescent reporters and multispectral ima- ging techniques to monitor complex protein–protein interactions (Hu and Kerppola, 2003; Waadt et al., 2008), protein and cellular movements (Hiraoka et al., 2002; Hoffman, 2005), transcriptional regulation due to multiple promoters (Cox et al., 2010) and for understanding morphological developments in in vitro or in Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ces Chemical Engineering Science 0009-2509/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ces.2013.06.021 n Corresponding author at: Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, rm: 4213 110 8th Street, Troy, NY 12180, United States. Tel.: +1 518 276 2138; fax: +1 518 276 3035. E-mail address: hahnj@rpi.edu (J. Hahn). 1 Present Address: Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, United States. Chemical Engineering Science 101 (2013) 191–198