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

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.

SUBMITTER: Takai A

PROVIDER: S-EPMC4394297 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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

Takai Akira A Nakano Masahiro M Saito Kenta K Haruno Remi R Watanabe Tomonobu M TM Ohyanagi Tatsuya T Jin Takashi T Okada Yasushi Y Nagai Takeharu T

Proceedings of the National Academy of Sciences of the United States of America 20150323 14

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 ...[more]

PMID: 25831507

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Project description:Rare-earth doped nanoparticles (RENPs) have been widely used for anti-counterfeiting and security applications due to their light frequency conversion features: they are excited at one wavelength, and they display spectrally narrow and distinguished luminescence peaks either at shorter wavelengths (i.e., frequency/energy upconversion) or at longer wavelengths (frequency/energy downconversion). RENPs with a downconversion (DC) photoluminescence (PL) in short-wave infrared (SWIR) spectral range (~1,000-1,700 nm) have recently been introduced to anti-counterfeiting applications, allowing for multilevel protection based on PL imaging through opaque layers, due to a lesser scattering of SWIR PL emission. However, as the number and spectral positions of the discrete PL bands exhibited by rare-earth ions are well-known, it is feasible to replicate luminescence spectra from RENPs, which results in a limited anti-counterfeiting security. Alternatively, lifetime of PL from RENPs can be used for encoding, as it can be finely tuned in broad temporal range (i.e., from microseconds to milliseconds) by varying type of dopants and their content in RENPs, along with the nanoparticle morphology and size. Nevertheless, the current approach to decoding and imaging the RENP luminescence lifetimes requires multiple steps and is highly time-consuming, precluding practical applications of PL lifetime encoding for anti-counterfeiting. Herein, we report the use of a rapid lifetime determination (RLD) technique to overcome this issue and introduce real-time imaging of SWIR PL lifetime for anti-counterfeiting applications. NaYF4:20% Yb, x% Er (x = 0, 2, 20, 80)@NaYF4 core@shell RENPs were synthesized and characterized, revealing DC PL in SWIR region, with maximum at ~1,530 nm and PL lifetimes ranging from 3.2 to 6 ms. Imaging of the nanoparticles with different lifetimes was performed by the developed time-gated imaging system engaging RLD method and the precise manipulation of the delay between the excitation pulses and camera gating windows. Moreover, it is shown that imaging and decrypting can be performed at a high rate (3-4 fps) in a cyclic manner, thus allowing for real-time temporal decoding. We believe that the demonstrated RLD-based fast PL lifetime imaging approach can be employed in other applications of photoluminescent RENPs.

Dataset Information

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

Publications

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

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