Analytical and Bioanalytical Chemistry

, Volume 406, Issue 23, pp 5735–5742 | Cite as

Dual-color bioluminescence imaging assay using green- and red-emitting beetle luciferases at subcellular resolution

  • Mayu Yasunaga
  • Yoshihiro NakajimaEmail author
  • Yoshihiro OhmiyaEmail author
Research Paper
Part of the following topical collections:
  1. Analytical Bioluminescence and Chemiluminescence


Bioluminescence imaging is widely used to monitor cellular events, including gene expression in vivo and in vitro. Moreover, recent advances in luciferase technology have made possible imaging at the single-cell level. To improve the bioluminescence imaging system, we have developed a dual-color imaging system in which the green-emitting luciferase from a Brazilian click beetle (Emerald Luc, ELuc) and the red-emitting luciferase from a railroad worm (Stable Luciferase Red, SLR) were used as reporters, which were localized to the peroxisome and the nucleus, respectively. We clearly captured simultaneously the subcellular localization of ELuc in the peroxisome and SLR in the nucleus of a single cell using a high-magnification objective lens with 3-min exposure time without binning using a combination of optical filters. Furthermore, to apply this system to quantitative time-lapse imaging, the activation of nuclear factor triggered by tumor necrosis factor α was measured using nuclear-targeted SLR and peroxisome-targeted ELuc as the test and internal control reporters, respectively. We successfully quantified the kinetics of activation of nuclear factor κB using nuclear-targeted SLR and the transcriptional change of the internal control promoter using peroxisome-targeted ELuc simultaneously in a single cell, and showed that the activation kinetics, including activation rate and amplitude, differed among cells. The results demonstrated that this imaging system can visualize the subcellular localization of reporters and track the expressions of two genes simultaneously at subcellular resolution.


Dual-color bioluminescence imaging Luciferase Nuclear factor κB Subcellular imaging Time-lapse imaging 



We thank T. Yamazaki from Toyobo for providing the NF-κB reporter vector. We also thank S. Kumata and M. Sasao from the National Institute of Advanced Industrial Science and Technology for excellent technical assistance. This study was supported by a Grant-in-Aid for Scientific Research (no. 21590266 to Y.N.) from the Japan Society for the Promotion of Science.

Supplementary material

216_2014_7981_MOESM1_ESM.docx (828 kb)
ESM 1 (DOCX 828 kb)
216_2014_7981_MOESM2_ESM.avi (1.1 mb)
ESM 2 (AVI 1.13 mb)
216_2014_7981_MOESM3_ESM.avi (2 mb)
ESM 3 (AVI 2.01 mb)

(AVI 1.51 mb)

216_2014_7981_MOESM5_ESM.avi (1.3 mb)
ESM 5 (AVI 1.29 mb)
216_2014_7981_MOESM6_ESM.avi (1.3 mb)
ESM 6 (AVI 1.29 mb)

(AVI 1.72 mb)


  1. 1.
    Naylor LH (1999) Reporter gene technology: the future looks bright. Biochem Pharmacol 58:749–757CrossRefGoogle Scholar
  2. 2.
    Greer LF 3rd, Szalay AA (2002) Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17:43–74CrossRefGoogle Scholar
  3. 3.
    Gross S, Piwnica-Worms D (2005) Spying on cancer: molecular imaging in vivo with genetically encoded reporters. Cancer Cell 7:5–15Google Scholar
  4. 4.
    Roda A, Guardigli M, Michelini E, Mirasoli M (2009) Bioluminescence in analytical chemistry and in vivo imaging. Trends Anal Chem 28:307–322CrossRefGoogle Scholar
  5. 5.
    Nakajima Y, Ohmiya Y (2010) Bioluminescence assays: multicolor luciferase assay, secreted luciferase assay and imaging luciferase assay. Expert Opin Drug Discov 5:835–849CrossRefGoogle Scholar
  6. 6.
    Welsh DK, Takahashi JS, Kay SA (2010) Suprachiasmatic nucleus: cell autonomy and network properties. Annu Rev Physiol 72:551–577CrossRefGoogle Scholar
  7. 7.
    Wilson T, Hastings JW (1998) Bioluminescence. Annu Rev Cell Dev Biol 14:197–230CrossRefGoogle Scholar
  8. 8.
    Gandelman O, Allue I, Bowers K, Cobbold P (1994) Cytoplasmic factors that affect the intensity and stability of bioluminescence from firefly luciferase in living mammalian cells. J Biolumin Chemilumin 9:363–371CrossRefGoogle Scholar
  9. 9.
    Ignowski JM, Schaffer DV (2004) Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells. Biotechnol Bioeng 86:827–834CrossRefGoogle Scholar
  10. 10.
    Luker KE, Luker GD (2008) Applications of bioluminescence imaging to antiviral research and therapy: multiple luciferase enzymes and quantitation. Antiviral Res 78:179–187CrossRefGoogle Scholar
  11. 11.
    Welsh DK, Kay SA (2005) Bioluminescence imaging in living organisms. Curr Opin Biotechnol 16:73–78CrossRefGoogle Scholar
  12. 12.
    Nakajima Y, Yamazaki T, Nishii S, Noguchi T, Hoshino H, Niwa K, Viviani VR, Ohmiya Y (2010) Enhanced beetle luciferase for high-resolution bioluminescence imaging. PLoS One 5:e10011CrossRefGoogle Scholar
  13. 13.
    Kitayama Y, Kondo T, Nakahira Y, Nishimura H, Ohmiya Y, Oyama T (2004) An in vivo dual-reporter system of cyanobacteria using two railroad-worm luciferases with different color emissions. Plant Cell Physiol 45:109–113CrossRefGoogle Scholar
  14. 14.
    Cevenini L, Michelini E, D'Elia M, Guardigli M, Roda A (2013) Dual-color bioluminescent bioreporter for forensic analysis: evidence of androgenic and anti-androgenic activity of illicit drugs. Anal Bioanal Chem 405:1035–1045CrossRefGoogle Scholar
  15. 15.
    Ogura R, Matsuo N, Wako N, Tanaka T, Ono S, Hiratsuka K (2005) Multi-color luciferase as reporters for monitoring transient gene expression in higher plants. Plant Biotechnol 22:151–155CrossRefGoogle Scholar
  16. 16.
    Nakajima Y, Ikeda M, Kimura T, Honma S, Ohmiya Y, Honma K (2004) Bidirectional role of orphan nuclear receptor RORα in clock gene transcriptions demonstrated by a novel reporter assay system. FEBS Lett 565:122–126CrossRefGoogle Scholar
  17. 17.
    Branchini BR, Southworth TL, Khattak NF, Michelini E, Roda A (2005) Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem 345:140–148CrossRefGoogle Scholar
  18. 18.
    Nakajima Y, Kimura T, Sugata K, Enomoto T, Asakawa A, Kubota H, Ikeda M, Ohmiya Y (2005) Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate. Biotechniques 38:891–894CrossRefGoogle Scholar
  19. 19.
    Branchini BR, Ablamsky DM, Murtiashaw MH, Uzasci L, Fraga H, Southworth TL (2007) Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem 361:253–262CrossRefGoogle Scholar
  20. 20.
    Davis RE, Zhang YQ, Southall N, Staudt LM, Austin CP, Inglese J, Auld DS (2007) A cell-based assay for IκBα stabilization using a two-color dual luciferase-based sensor. Assay Drug Dev Technol 5:85–103CrossRefGoogle Scholar
  21. 21.
    Michelini E, Cevenini L, Mezzanotte L, Ablamsky D, Southworth T, Branchini B, Roda A (2008) Spectral-resolved gene technology for multiplexed bioluminescence and high-content screening. Anal Chem 80:260–267CrossRefGoogle Scholar
  22. 22.
    Noguchi T, Ikeda M, Ohmiya Y, Nakajima Y (2008) Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting luciferases. BMC Biotechnol 8:40CrossRefGoogle Scholar
  23. 23.
    Kafi AKM, Hattori M, Misawa N, Ozawa T (2011) Dual-color bioluminescence analysis for quantitatively monitoring G-protein-coupled receptor and β-arrestin interactions. Pharmaceuticals 4:457–469CrossRefGoogle Scholar
  24. 24.
    Saito R, Hirakawa S, Ohara H, Yasuda M, Yamazaki T, Nishii S, Aiba S (2011) Nickel differentially regulates NFAT and NF-κB activation in T cell signaling. Toxicol Appl Pharmacol 254:245–255CrossRefGoogle Scholar
  25. 25.
    Takahashi T, Kimura Y, Saito R, Nakajima Y, Ohmiya Y, Yamasaki K, Aiba S (2011) An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8. Toxicol Sci 124:359–369CrossRefGoogle Scholar
  26. 26.
    Noguchi T, Michihata T, Nakamura W, Takumi T, Shimizu R, Yamamoto M, Ikeda M, Ohmiya Y, Nakajima Y (2010) Dual-color luciferase mouse directly demonstrates coupled expression of two clock genes. Biochemistry 49:8053–8061CrossRefGoogle Scholar
  27. 27.
    Noguchi T, Ikeda M, Ohmiya Y, Nakajima Y (2012) A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands. PLoS One 7:e37093CrossRefGoogle Scholar
  28. 28.
    Naik S, Piwnica-Worms D (2007) Real-time imaging of β-catenin dynamics in cells and living mice. Proc Natl Acad Sci U S A 104:17465–17470CrossRefGoogle Scholar
  29. 29.
    Hida N, Awais M, Takeuchi M, Ueno N, Tashiro M, Takagi C, Singh T, Hayashi M, Ohmiya Y, Ozawa T (2009) High-sensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases. PLoS One 4:e5868CrossRefGoogle Scholar
  30. 30.
    Mezzanotte L, Que I, Kaijzel E, Branchini B, Roda A, Lowik C (2011) Sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase. PLoS One 6:e19277CrossRefGoogle Scholar
  31. 31.
    Mezzanotte L, Aswendt M, Tennstaedt A, Hoeben R, Hoehn M, Lowik C (2013) Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain. Contrast Media Mol Imaging 8:505–513CrossRefGoogle Scholar
  32. 32.
    Kwon H, Enomoto T, Shimogawara M, Yasuda K, Nakajima Y, Ohmiya Y (2010) Bioluminescence imaging of dual gene expression at the single-cell level. Biotechniques 48:460–462CrossRefGoogle Scholar
  33. 33.
    Niwa Y, Shimojo H, Isomura A, Gonzalez A, Miyachi H, Kageyama R (2011) Different types of oscillations in Notch and Fgf signaling regulate the spatiotemporal periodicity of somitogenesis. Genes Dev 25:1115–1120CrossRefGoogle Scholar
  34. 34.
    Imayoshi I, Isomura A, Harima Y, Kawaguchi K, Kori H, Miyachi H, Fujiwara T, Ishidate F, Kageyama R (2013) Oscillatory control of factors determining multipotency and fate in mouse neural progenitors. Science 342:1203–1208CrossRefGoogle Scholar
  35. 35.
    Viviani VR, Silva AC, Perez GL, Santelli RV, Bechara EJ, Reinach FC (1999) Cloning and molecular characterization of the cDNA for the Brazilian larval click-beetle Pyrearinus termitilluminans luciferase. Photochem Photobiol 70:254–260CrossRefGoogle Scholar
  36. 36.
    Viviani VR, Bechara EJ, Ohmiya Y (1999) Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures. Biochemistry 38:8271–8279CrossRefGoogle Scholar
  37. 37.
    Niwa K, Ichino Y, Ohmiya Y (2010) Quantum yield measurements of firefly bioluminescence reactions using a commercial luminometer. Chem Lett 39:291–293CrossRefGoogle Scholar
  38. 38.
    Baker RG, Hayden MS, Ghosh S (2011) NF-κB, inflammation, and metabolic disease. Cell Metab 13:11–22CrossRefGoogle Scholar
  39. 39.
    Tornatore L, Thotakura AK, Bennett J, Moretti M, Franzoso G (2012) The nuclear factor kappa B signaling pathway: integrating metabolism with inflammation. Trends Cell Biol 22:557–566CrossRefGoogle Scholar
  40. 40.
    Ling J, Kumar R (2012) Crosstalk between NFκB and glucocorticoid signaling: a potential target of breast cancer therapy. Cancer Lett 322:119–126CrossRefGoogle Scholar
  41. 41.
    Kwon HJ, Ohmiya Y, Honma KI, Honma S, Nagai T, Saito K, Yasuda K (2012) Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis. Cell Death Dis 3:e278CrossRefGoogle Scholar
  42. 42.
    Kwon HJ, Ohmiya Y, Yasuda K (2012) Dual-color system for simultaneously monitoring intracellular Ca2+ and ATP dynamics. Anal Biochem 430:45–47CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Health Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)TakamatsuJapan
  2. 2.DAILAB, Biomedical Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan

Personalised recommendations