Upconversion luminescence imaging of cells and small animals

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.

[1]  Wei Feng,et al.  Blue-emissive upconversion nanoparticles for low-power-excited bioimaging in vivo. , 2012, Journal of the American Chemical Society.

[2]  Jun Lin,et al.  Hydrothermal Synthesis of Lanthanide Fluorides LnF3 (Ln = La to Lu) Nano-/Microcrystals with Multiform Structures and Morphologies , 2008 .

[3]  Ya-Wen Zhang,et al.  High-quality sodium rare-earth fluoride nanocrystals: controlled synthesis and optical properties. , 2006, Journal of the American Chemical Society.

[4]  G. Chow,et al.  Synthesis of Hexagonal‐Phase NaYF4:Yb,Er and NaYF4:Yb,Tm Nanocrystals with Efficient Up‐Conversion Fluorescence , 2006 .

[5]  Victoria J Allan,et al.  Light Microscopy Techniques for Live Cell Imaging , 2003, Science.

[6]  Xu-Hua Wang,et al.  High-Fidelity Hydrophilic Probe for Two-Photon Fluorescence Lysosomal Imaging , 2010, Journal of the American Chemical Society.

[7]  Wei Feng,et al.  Cubic sub-20 nm NaLuF(4)-based upconversion nanophosphors for high-contrast bioimaging in different animal species. , 2012, Biomaterials.

[8]  F. Castellano,et al.  eview hoton upconversion based on sensitized triplet – triplet annihilation , 2010 .

[9]  Gregory S Harms,et al.  Upconverting nanoparticles for nanoscale thermometry. , 2011, Angewandte Chemie.

[10]  Hongjie Dai,et al.  Ag2S quantum dot: a bright and biocompatible fluorescent nanoprobe in the second near-infrared window. , 2012, ACS nano.

[11]  R. Weissleder,et al.  Imaging in the era of molecular oncology , 2008, Nature.

[12]  F. Auzel.  Upconversion and anti-Stokes processes with f and d ions in solids. , 2004, Chemical reviews.

[13]  Yaron Silberberg,et al.  Coherent quantum control of two-photon transitions by a femtosecond laser pulse , 1998, Nature.

[14]  M. Haase,et al.  Lanthanide-Doped NaYF4 Nanocrystals in Aqueous Solution Displaying Strong Up-Conversion Emission , 2007 .

[15]  Louis A. Cuccia,et al.  Synthesis of colloidal upconverting NaYF4 nanocrystals doped with Er3+, Yb3+ and Tm3+, Yb3+ via thermal decomposition of lanthanide trifluoroacetate precursors. , 2006, Journal of the American Chemical Society.

[16]  Qing Peng,et al.  A general strategy for nanocrystal synthesis , 2005, Nature.

[17]  Yuan Gao,et al.  Water-soluble NaYF4:Yb/Er upconversion nanophosphors: Synthesis, characteristics and application in bioimaging , 2010 .

[18]  Eva M Sevick-Muraca,et al.  Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents. , 2002, Current opinion in chemical biology.

[19]  Chun-Hua Yan,et al.  Bioimaging and toxicity assessments of near-infrared upconversion luminescent NaYF4:Yb,Tm nanocrystals. , 2011, Biomaterials.

[20]  Paras N. Prasad,et al.  (α-NaYbF4:Tm(3+))/CaF2 core/shell nanoparticles with efficient near-infrared to near-infrared upconversion for high-contrast deep tissue bioimaging. , 2012, ACS nano.

[21]  Renren Deng,et al.  Tuning upconversion through energy migration in core-shell nanoparticles. , 2011, Nature materials.

[22]  Qian Liu,et al.  A general strategy for biocompatible, high-effective upconversion nanocapsules based on triplet-triplet annihilation. , 2013, Journal of the American Chemical Society.

[23]  Fuyou Li,et al.  High contrast upconversion luminescence targeted imaging in vivo using peptide-labeled nanophosphors. , 2009, Analytical chemistry.

[24]  Felix N. Castellano,et al.  Photon upconversion based on sensitized triplet-triplet annihilation , 2010 .

[25]  Zhuang Liu,et al.  Upconversion nanophosphors for small-animal imaging. , 2012, Chemical Society reviews.

[26]  Zhigang Chen,et al.  Laser scanning up-conversion luminescence microscopy for imaging cells labeled with rare-earth nanophosphors. , 2009, Analytical chemistry.

[27]  Tymish Y. Ohulchanskyy,et al.  High contrast in vitro and in vivo photoluminescence bioimaging using near infrared to near infrared up-conversion in Tm3+ and Yb3+ doped fluoride nanophosphors. , 2008, Nano letters.

[28]  Helmut Schäfer,et al.  Upconverting nanoparticles. , 2011, Angewandte Chemie.

[29]  Wei Feng,et al.  Sub-10 nm hexagonal lanthanide-doped NaLuF4 upconversion nanocrystals for sensitive bioimaging in vivo. , 2011, Journal of the American Chemical Society.

[30]  Dan Wang,et al.  Using 915 nm laser excited Tm³+/Er³+/Ho³+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation. , 2011, ACS nano.

[31]  En Ma,et al.  Amine-functionalized lanthanide-doped zirconia nanoparticles: optical spectroscopy, time-resolved fluorescence resonance energy transfer biodetection, and targeted imaging. , 2012, Journal of the American Chemical Society.

[32]  Yong Zhang,et al.  Upconversion fluorescence imaging of cells and small animals using lanthanide doped nanocrystals. , 2008, Biomaterials.

[33]  Eric C. Carnes,et al.  Mesoporous silica nanoparticle nanocarriers: biofunctionality and biocompatibility. , 2013, Accounts of chemical research.

[34]  Jianzhang Zhao,et al.  Triplet Photosensitizers: From Molecular Design to Applications , 2013 .

[35]  Fuyou Li,et al.  Multimodal-luminescence core-shell nanocomposites for targeted imaging of tumor cells. , 2009, Chemistry.

[36]  Muthu Kumara Gnanasammandhan,et al.  In vivo photodynamic therapy using upconversion nanoparticles as remote-controlled nanotransducers , 2012, Nature Medicine.

[37]  Qingyu Zhang,et al.  Rate equation model analysis on the infrared and upconversion emission of Er/Yb co-doped borate-silicate glass , 2010 .