Spectral Unmixing of Multicolored Bioluminescence Emitted from Heterogeneous Biological Sources

• Published in: Analytical chemistry (Washington) Vol. 78; no. 5; pp. 1520 - 1527
• Main Authors: Gammon, Seth T, Leevy, W. Matthew, Gross, Shimon, Gokel, George W, Piwnica-Worms, David
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.03.2006
• Subjects: Analytical chemistry; Cell-Free System; Cells; Chemistry; Color; Diagnostic Imaging - instrumentation; Diagnostic Imaging - methods; Exact sciences and technology; Genes, Reporter; HeLa Cells; Humans; I-kappa B Kinase - analysis; Luciferases - analysis; Luciferases - genetics; Luminescence; Protein Processing, Post-Translational; Proteins; Recombinant Fusion Proteins - analysis; Renilla; Software; Transcription, Genetic; Transfection; Bioluminescence; Deconvolution; Biochemical analysis; Chemical analysis; CCD camera; Feasibility; Software; Overlap; Protein
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac051999h

A wide variety of bioluminescent luciferase proteins are available for use in transcriptional or biochemical reporter assays. However, spectral overlap normally prevents them from being monitored simultaneously. To address this problem, a Java plug-in for ImageJ was written to deconvolute bioluminescent images composed of signals from multiple luciferases. The methodology was validated by testing the program with both simulated and real luciferase images. Bioluminescent images were acquired using a CCD camera equipped with optical filters, and the images were deconvoluted using the ImageJ plug-in. HeLa cells were transfected with either click beetle red luciferase (CBR), click beetle green luciferase (CBG99), or Renilla luciferase (Rluc), and mixed lysates were imaged in varying proportions in a 96-well plate to biochemically validate the methodology. After spectral deconvolution, the predicted, pure luciferase signals could be recovered with maximal cross-talk errors of ±1.5%. In addition, live cells expressing CBR, CBG99, and Rluc were simultaneously imaged and deconvoluted in 96-well plates to demonstrate the feasibility of applying this methodology to high-throughput applications. Finally, multicolor transcriptional and posttranslational modification reporters were simultaneously imaged and shown to deconvolute normalized IκΒ kinase activity in longitudinal assays. Thus, our software provided a rapid, simple, and accurate method for simultaneously measuring multiple bioluminescent reporters in living cells.