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Upconversion luminescence imaging of cells and small animals

Nature Protocols volume 8, pages 2033–2044 (2013)Cite this article

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Abstract

Upconversion luminescence (UCL) is an anti-Stokes process whereby low-energy photons are converted to higher-energy ones. UCL imaging for cells and animal tissues has attracted substantial attention in recent years because of the unique abilities of upconversion materials, which can minimize the background interference from the autofluorescence of biosamples and enhance tissue penetration. This protocol describes a step-by-step guide for the fabrication of UCL probes, including lanthanide-based upconversion nanoparticles (Ln-UCNPs) with a particle size of ∼20 nm (NaYF4/NaLuF4: Yb, Er/Tm) and triplet-triplet annihilation-based UCNPs (TTA-UCNPs) with a particle size of ∼10 nm (palladium octaethylporphyrin as sensitizer and 9,10-diphenylanthracene as annihilator). We also describe the characterization of the UCL nanoprobes (via transmission electron microscopy and UCL emission spectroscopy) and functionalization (via silica coating and ligand exchange), as well as applications for UCL bioimaging of living cells (HeLa cells) and small animals (nude mice and Kunming mice). The setup of a laser-scanning UCL microscope and a UCL imaging system is also presented. Compared with a normal imaging setup, we adopted longer-wavelength excitation lasers and short-pass filters. The synthesis of hydrophilic UCNP for application in UCL bioimaging requires ∼15 d.

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Figure 1: Schematic representation of the conventional luminescence and upconversion luminescence processes.
Figure 2: Schematic layout of the setup for measuring the UCL emission spectrum.
Figure 3: Schematic layout for the laser scanning UCL confocal microscope setup for UCL imaging.
Figure 4: Diagram depicting the experimental setup for the small-animal UCL imaging system.
Figure 5: Characterization of UCNPs and their applications in cell imaging.
Figure 6: SNR analysis of UCL imaging in living cells.
Figure 7: UCL photostability in living cells.
Figure 8: UCL lymphatic imaging.
Figure 9: UCL cell tracking.

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Acknowledgements

HeLa lines were provided by the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Chinese Academy of Science. This study was supported by grants from the National Science Foundation of China (21231004), Ministry of Science and Technology, China (2013CB733700, 2012CB932403 and 2011AA03A407) and Science and Technology Commission of Shanghai Municipality (11XD1400300 and 12JC1401300).

Author information

Authors and Affiliations

  1. Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Institutes of Biomedical Sciences, and Concerted Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai, P. R. China

    Qian Liu, Wei Feng, Tianshe Yang, Tao Yi & Fuyou Li

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Contributions

Q.L., W.F. and T. Yang designed the protocol and carried out the experiments. T. Yi and F.L. designed and supervised the project. Q.L., W.F. and F.L. wrote the manuscript.

Corresponding author

Correspondence to Fuyou Li.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Upconversion mechanism of the Yb,Er/Tm co-doped Ln-UCNPs.

Under excitation at 980 nm, an electron of Yb3+ is excited from the 2F7/2 to the 2F5/2 level. The energy may be transferred to Er3+/Tm3+ nonradiatively to excite it to the corresponding excited level. For Yb,Er co-doped Ln-UCNPs, the emission bands at 520, 540, and 654 nm may be assigned to 2H11/2→4I15/2, 4S3/2→4I15/2, and 4F9/2→4I15/2 transitions of Er3+. For Yb,Tm co-doped Ln-UCNPs, the emission bands at 365, 451, 481, 646 and 800 nm may be assigned to 1D2→3H6, 1D2→3F4, 1G4→3H6, 1G4→3F4, and 3H4→3H6, respectively.

Supplementary Figure 2 Generalized energy-level diagram of the TTA-based upconversion processes.

Generalized energy-level diagram of the upconversion processes between the triplet state of the sensitizer molecule and the triplet annihilator molecule leading to singlet delayed fluorescence. Colored solid lines represent radiative processes. 1S and 3T are the singlet state and the triplet state of sensitizer or annihilator, respectively. GS is the ground state, ISC denotes intersystem crossing, TTET denotes triplet-triplet energy transfer, and TTA denotes triplet-triplet annihilation.

Supplementary Figure 3 Photographs of laser-scanning UCL confocal microscopy.

Supplementary Figure 4 Photographs of UCL imaging in vivo system.

Supplementary Figure 5 UCL image of living HeLa cells incubated with silica-modified Ln-UCNPs.

(a) UCL image of living HeLa cells incubated with silica-modified Ln-UCNPs when excited with 980 nm (λem = 600 nm~700 nm). (b) Overlay of bright-field image and UCL images; the power in the focal planes is 19 mW.

Supplementary Figure 6 Fluorescence image of living HeLa cells incubated with TTA-UCNPs.

(a) Conventional fluorescence image of living HeLa cells incubated with TTA-UCNPs, excited at 405 nm; emission was collected at 420-480 nm. (b) Overlay of bright-field image, luminescence images (a) and a TTA-UCL image.

Supplementary information

Supplementary Figure 1

Upconversion mechanism of the Yb,Er/Tm co-doped Ln-UCNPs. (PDF 128 kb)

Supplementary Figure 2

Generalized energy-level diagram of the TTA-based upconversion processes. (PDF 80 kb)

Supplementary Figure 3

Photographs of laser-scanning UCL confocal microscopy. (PDF 105 kb)

Supplementary Figure 4

Photographs of UCL imaging in vivo system. (PDF 115 kb)

Supplementary Figure 5

UCL image of living HeLa cells incubated with silica-modified Ln-UCNPs. (PDF 34 kb)

Supplementary Figure 6

Fluorescence image of living HeLa cells incubated with TTA-UCNPs. (PDF 30 kb)

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Liu, Q., Feng, W., Yang, T. et al. Upconversion luminescence imaging of cells and small animals. Nat Protoc 8, 2033–2044 (2013). https://doi.org/10.1038/nprot.2013.114

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