Spectral unmixing of multicolored bioluminescence emitted from heterogeneous biological sources
Spectral unmixing of multicolored bioluminescence emitted from heterogeneous biological sources
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DOI：
10.1021/ac051999h
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发表时间：
2006-03-01
影响因子：
7.4
通讯作者：
Piwnica-Worms, D
中科院分区：
文献类型：
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作者：
Gammon, STM;Leevy, WM;Piwnica-Worms, D
多种生物发光荧光素酶蛋白可用于转录或生化报告分析。然而，光谱重叠通常会阻止它们同时监测。为了解决这个问题，编写了 ImageJ 的 Java 插件来对由多种荧光素酶信号组成的生物发光图像进行解卷积。通过使用模拟和真实荧光素酶图像测试程序来验证该方法。使用配备滤光片的 CCD 相机获取生物发光图像，并使用 ImageJ 插件对图像进行解卷积。 HeLa 细胞用点击甲虫红色荧光素酶 (CBR)、点击甲虫绿色荧光素酶 (CBG99) 或海肾荧光素酶 (Rluc) 转染，并在 96 孔板中以不同比例对混合裂解物进行成像，以对方法进行生化验证。光谱解卷积后，可以恢复预测的纯荧光素酶信号，最大串扰误差为 1.5%。此外，在 96 孔板中同时对表达 CBR、CBG99 和 Rluc 的活细胞进行成像和解卷积，以证明将该方法应用于高通量应用的可行性。最后，多色转录和翻译后修饰报告基因同时成像，并显示可在纵向测定中对标准化 I kappa B 激酶活性进行解卷积。因此，我们的软件提供了一种快速、简单且准确的方法，用于同时测量活细胞中的多个生物发光报告基因。
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 I kappa B kinase activity in longitudinal assays. Thus, our software provided a rapid, simple, and accurate method for simultaneously measuring multiple bioluminescent reporters in living cells.