Spectral Unmixing of Multicolored Bioluminescence Emitted from Heterogeneous Biological Sources

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• 作者：Seth T. Gammon ; W. Matthew Leevy ; Shimon Gross ; George W. Gokel ; and David Piwnica-Worms
• 刊名：Analytical Chemistry
• 出版年：2006
• 出版时间：March 1, 2006
• 年：2006
• 卷：78
• 期：5
• 页码：1520 - 1527
• 全文大小：505K
• 年卷期：v.78,no.5(March 1, 2006)
• ISSN：1520-6882

文摘

A wide variety of bioluminescent luciferase proteins areavailable for use in transcriptional or biochemical reporterassays. However, spectral overlap normally prevents themfrom being monitored simultaneously. To address thisproblem, a Java plug-in for ImageJ was written to deconvolute bioluminescent images composed of signals frommultiple luciferases. The methodology was validated bytesting the program with both simulated and real luciferase images. Bioluminescent images were acquiredusing a CCD camera equipped with optical filters, and theimages were deconvoluted using the ImageJ plug-in. HeLacells were transfected with either click beetle red luciferase (CBR), click beetle green luciferase (CBG99), orRenilla luciferase (Rluc), and mixed lysates were imagedin varying proportions in a 96-well plate to biochemicallyvalidate the methodology. After spectral deconvolution,the predicted, pure luciferase signals could be recoveredwith maximal cross-talk errors of ±1.5%. In addition, livecells expressing CBR, CBG99, and Rluc were simultaneously imaged and deconvoluted in 96-well plates todemonstrate the feasibility of applying this methodologyto high-throughput applications. Finally, multicolor transcriptional and posttranslational modification reporterswere simultaneously imaged and shown to deconvolutenormalized I kinase activity in longitudinal assays.Thus, our software provided a rapid, simple, and accuratemethod for simultaneously measuring multiple bioluminescent reporters in living cells.