Tunable lifetime multiplexing using luminescent nanocrystals

Journal name:
Nature Photonics
Year published:
Published online


Optical multiplexing plays an important role in applications such as optical data storage1, document security2, molecular probes3, 4 and bead assays for personalized medicine5. Conventional fluorescent colour coding is limited by spectral overlap and background interference, restricting the number of distinguishable identities. Here, we show that tunable luminescent lifetimes τ in the microsecond region can be exploited to code individual upconversion nanocrystals. In a single colour band, one can generate more than ten nanocrystal populations with distinct lifetimes ranging from 25.6 µs to 662.4 µs and decode their well-separated lifetime identities, which are independent of both colour and intensity. Such ‘τ-dots’ potentially suit multichannel bioimaging, high-throughput cytometry quantification, high-density data storage, as well as security codes to combat counterfeiting. This demonstration extends the optical multiplexing capability by adding the temporal dimension of luminescent signals, opening new opportunities in the life sciences, medicine and data security.

At a glance


  1. Lifetime tuning scheme and time-resolved confocal images for NaYF4:Yb,Tm upconversion nanocrystals.
    Figure 1: Lifetime tuning scheme and time-resolved confocal images for NaYF4:Yb,Tm upconversion nanocrystals.

    Each pixel was excited for 200 µs, followed by a delayed detection window of up to 3.8 ms to record its time-gated luminescence decay (40 ms exposure time to allow 10 times integration). The colour tone (hue) for each pixel represents its lifetime value decoded from the decay curve. The nanocrystals in the images from left to right have Tm doping concentrations of 4, 2, 1, 0.5 and 0.2 mol%, respectively, as well as 20 mol% Yb dopants.

  2. Results for [tau]-dots-labelled Giardia cysts measured by the time-resolved scanning cytometry system.
    Figure 2: Results for τ-dots-labelled Giardia cysts measured by the time-resolved scanning cytometry system.

    a,b, Lifetime histograms obtained from cysts labelled with different lifetime-encoded τ-dots (Yb/Tm co-doping concentration (mol%:mol%) of 20:1 for a and 20:4 for b). The scanning cytometry allows retrieval of each individual target cyst for luminescence as well as bright-field imaging confirmation. c, Typical recorded luminescence images for the same cyst under 4 h continuous laser excitation. All images were captured with a 100 ms exposure time.

  3. Concept of [tau]-dots-encoded microspheres as the lifetime multiplexing suspension arrays.
    Figure 3: Concept of τ-dots-encoded microspheres as the lifetime multiplexing suspension arrays.

    a, The synthesized monodispersed Tm upconversion nanocrystals can be embedded into the shell of porous microspheres, which can be decoded by the time-resolved scanning cytometry system, for example. b,c, Typical TEM image of the nanocrystals (b) and SEM image of a microsphere incorporating the nanocrystals (c).

  4. Results for [tau]-dots-encoded populations of microspheres carrying unique lifetime identities.
    Figure 4: Results for τ-dots-encoded populations of microspheres carrying unique lifetime identities.

    a, The mechanism of upconversion energy transfer, by adjusting the co-dopant concentration of the sensitizer/emitter, can generate eight lifetime populations of microspheres in the Tm blue-emission band. Symbols α and β represent cubic and hexagonal crystal phases, respectively. The numeral besides each histogram is the mean lifetime ± lifetime CV from Gaussian distribution fitting. The blocks in the axis above represent the lifetime resources (±3σ) occupied by each population. The open spaces suggest more populations could be engineered. b, Two-dimensional (intensity versus lifetime) scattered plots showing that all lifetime populations are independent of the intensities of individual microcarriers.

  5. Demonstration of lifetime-encoded document security and photonic data storage.
    Figure 5: Demonstration of lifetime-encoded document security and photonic data storage.

    ac, Three overlapping patterns are printed with different Tm τ-dots: (CYb:CTm) 20:4 for the ‘Macquarie University’ logo, 20:1 for the Sydney Opera House image, and 20:0.5 for the Sydney Harbour Bridge image. Intensity-based luminescence imaging only gives a complex picture (a), but time-resolved scanning separates the patterns based on the lifetime components of every pixel (b), so that genuine multiplexing information contained in the same overlapping space of the document can be decoded (c; pseudocolour is used to indicate the luminescence lifetime for each pixel). Scale bars (all images), 5 mm.


  1. Zijlstra, P., Chon, J. W. M. & Gu, M. Five-dimensional optical recording mediated by surface plasmons in gold nanorods. Nature 459, 410413 (2009).
  2. Jeevan, M. M. et al. Security printing of covert quick response codes using upconverting nanoparticle inks. Nanotechnology 23, 395201 (2012).
  3. Li, Y. G., Cu, Y. T. H. & Luo, D. Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes. Nature Biotechnol. 23, 885889 (2005).
  4. Lin, C. X. et al. Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA. Nature Chem. 4, 832839 (2012).
  5. Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834838 (2005).
  6. Schena, M., Shalon, D., Davis, R. W. & Brown, P. O. Quantitative monitoring of gene-expression patterns with a complementary-DNA microarray. Science 270, 467470 (1995).
  7. Thomson, J. M., Parker, J., Perou, C. M. & Hammond, S. M. A custom microarray platform for analysis of microRNA gene expression. Nature Methods 1, 4753 (2004).
  8. Nicewarner-Pena, S. R. et al. Submicrometer metallic barcodes. Science 294, 137141 (2001).
  9. Pregibon, D. C., Toner, M. & Doyle, P. S. Multifunctional encoded particles for high-throughput biomolecule analysis. Science 315, 13931396 (2007).
  10. Braeckmans, K. et al. Encoding microcarriers by spatial selective photobleaching. Nature Mater. 2, 169173 (2003).
  11. Han, M. Y., Gao, X. H., Su, J. Z. & Nie, S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nature Biotechnol. 19, 631635 (2001).
  12. Wang, F. et al. Tuning upconversion through energy migration in core-shell nanoparticles. Nature Mater. 10, 968973 (2011).
  13. Cunin, F. et al. Biomolecular screening with encoded porous-silicon photonic crystals. Nature Mater. 1, 3941 (2002).
  14. Cao, Y. W. C., Jin, R. C. & Mirkin, C. A. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 297, 15361540 (2002).
  15. Bendall, S. C. et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687696 (2011).
  16. Lander, E. S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860921 (2001).
  17. Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281297 (2004).
  18. Pawson, T. & Nash, P. Assembly of cell regulatory systems through protein interaction domains. Science 300, 445452 (2003).
  19. Nicholson, J. K. & Lindon, J. C. Systems biology—metabonomics. Nature 455, 10541056 (2008).
  20. Van't Veer, L. J. & Bernards, R. Enabling personalized cancer medicine through analysis of gene-expression patterns. Nature 452, 564570 (2008).
  21. Li, X., Lan, T.-H., Tien, C.-H. & Gu, M. Three-dimensional orientation-unlimited polarization encryption by a single optically configured vectorial beam. Nature Commun. 3, 998 (2012).
  22. Perfetto, S. P., Chattopadhyay, P. K. & Roederer, M. Seventeen-colour flow cytometry: unravelling the immune system. Nature Rev. Immunol. 4, 648655 (2004).
  23. Cui, H. H., Valdez, J. G., Steinkamp, J. A. & Crissman, H. A. Fluorescence lifetime-based discrimination and quantification of cellular DNA and RNA with phase-sensitive flow cytometry. Cytometry A 52A, 4655 (2003).
  24. Watson, D. A. et al. A flow cytometer for the measurement of Raman spectra. Cytometry A 73A, 119128 (2008).
  25. Gnach, A. & Bednarkiewicz, A. Lanthanide-doped up-converting nanoparticles: merits and challenges. Nano Today 7, 532563 (2012).
  26. Wang, F. et al. Simultaneous phase and size control of upconversion nanocrystals through lanthanide doping. Nature 463, 10611065 (2010).
  27. Lu, Y., Xi, P., Piper, J. A., Huo, Y. & Jin, D. Time-gated orthogonal scanning automated microscopy (OSAM) for high-speed cell detection and analysis. Sci. Rep. 2, 837 (2012).
  28. Yang, L., Tran, D. K. & Wang, X. BADGE, BeadsArray for the detection of gene expression, a high-throughput diagnostic bioassay. Genome Res. 11, 18881898 (2001).
  29. Yurkovetsky, Z. R. et al. Multiplex analysis of serum cytokines in melanoma patients treated with interferon-alpha 2b. Clin. Cancer Res. 13, 24222428 (2007).
  30. Wang, F., Wang, J. & Liu, X. Direct evidence of a surface quenching effect on size-dependent luminescence of upconversion nanoparticles. Angew. Chem. Int. Ed. 49, 74567460 (2010).
  31. Zhao, J. et al. Upconversion luminescence with tunable lifetime in NaYF4:Yb,Er nanocrystals: role of nanocrystal size. Nanoscale 5, 944952 (2013).
  32. Bastiaens, P. I. H. & Squire, A. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9, 4852 (1999).
  33. Heilemann, M. et al. High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy. Anal. Chem. 74, 35113517 (2002).

Download references

Author information


  1. Advanced Cytometry Laboratories, MQ Photonics Research Centre and MQ BioFocus Research Centre, Macquarie University, Sydney, New South Wales 2109, Australia

    • Yiqing Lu,
    • Jiangbo Zhao,
    • Run Zhang,
    • Yujia Liu,
    • Deming Liu,
    • Ewa M. Goldys,
    • Jie Lu,
    • Yu Shi,
    • James A. Piper &
    • Dayong Jin
  2. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia

    • Run Zhang,
    • Anwar Sunna,
    • Jie Lu,
    • Yu Shi &
    • Dayong Jin
  3. Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China

    • Yujia Liu,
    • Xusan Yang &
    • Peng Xi
  4. School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China

    • Yujia Liu
  5. Newport Instruments, 3345 Hopi Place, San Diego, California 92117-3516, USA

    • Robert C. Leif
  6. Department of Electronic Engineering, Tsinghua University, Beijing 100084, China

    • Yujing Huo
  7. Olympus Australia, 82 Waterloo Road, North Ryde, New South Wales 2113, Australia

    • Jian Shen
  8. Purdue University Cytometry Laboratories, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907, USA

    • J. Paul Robinson &
    • Dayong Jin


D.J., J.A.P., R.C.L. and J.P.R. conceived the project. D.J. designed the experiments and supervised the research. Y.Lu, J.Z. and D.J. were primarily responsible for data collection and analysis. Y.Lu, E.M.G. and D.J. prepared figures and wrote the main manuscript text. Y.Lu, J.Z., R.Z., D.L. and D.J. were primarily responsible for the Supplementary Information. All authors contributed to data analysis, discussions and manuscript preparation.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary information (2,764 KB)

    Supplementary information

Additional data