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Proc Natl Acad Sci U S A. 2015 Apr 7;112(14):4352-6. doi: 10.1073/pnas.1418468112. Epub 2015 Mar 23.

Expanded palette of Nano-lanterns for real-time multicolor luminescence imaging.

Author information

1
Laboratories for Cell Polarity Regulation.
2
The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan; and.
3
Comprehensive Bioimaging, PRESTO, Japan Science and Technology Agency, Tokyo 102-0075, Japan.
4
Nano-Bio Probes, and.
5
Laboratories for Cell Polarity Regulation, ng1@sanken.osaka-u.ac.jp y.okada@riken.jp.
6
The Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan; and PRESTO, Japan Science and Technology Agency, Tokyo 102-0075, Japan Cell Dynamics Observation, Quantitative Biology Center, RIKEN, Osaka 565-0874, Japan; ng1@sanken.osaka-u.ac.jp y.okada@riken.jp.

Abstract

Fluorescence live imaging has become an essential methodology in modern cell biology. However, fluorescence requires excitation light, which can sometimes cause potential problems, such as autofluorescence, phototoxicity, and photobleaching. Furthermore, combined with recent optogenetic tools, the light illumination can trigger their unintended activation. Because luminescence imaging does not require excitation light, it is a good candidate as an alternative imaging modality to circumvent these problems. The application of luminescence imaging, however, has been limited by the two drawbacks of existing luminescent protein probes, such as luciferases: namely, low brightness and poor color variants. Here, we report the development of bright cyan and orange luminescent proteins by extending our previous development of the bright yellowish-green luminescent protein Nano-lantern. The color change and the enhancement of brightness were both achieved by bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein. The brightness of these cyan and orange Nano-lanterns was ∼20 times brighter than wild-type Renilla luciferase, which allowed us to perform multicolor live imaging of intracellular submicron structures. The rapid dynamics of endosomes and peroxisomes were visualized at around 1-s temporal resolution, and the slow dynamics of focal adhesions were continuously imaged for longer than a few hours without photobleaching or photodamage. In addition, we extended the application of these multicolor Nano-lanterns to simultaneous monitoring of multiple gene expression or Ca(2+) dynamics in different cellular compartments in a single cell.

KEYWORDS:

bioluminescence resonance energy transfer; live imaging; luciferase; luminescence imaging; luminescent indicator

PMID:
25831507
PMCID:
PMC4394297
DOI:
10.1073/pnas.1418468112
[Indexed for MEDLINE]
Free PMC Article

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