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Ultrasensitive optical imaging with lanthanide lumiphores


In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identified three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcame these limitations with a new lanthanide imaging modality, transreflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over that of conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.

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Figure 1: Time-resolved lanthanide detection and LRET enhancement.
Figure 2: LRET-enhanced time-resolved imaging of lanthanide-functionalized beads.
Figure 3: Optics and lanthanide photoluminescence overlap temporally and spectrally.
Figure 4: QSL transreflected illumination overcomes optics-derived photoluminescence.
Figure 5: QSL excitation dramatically increases lanthanide excitation rates.
Figure 6: trLRET enables ultrasensitive lanthanide imaging in vivo.


  1. 1

    Grimm, J.B., Heckman, L.M. & Lavis, L.D. The chemistry of small-molecule fluorogenic probes. Prog. Mol. Biol. Transl. Sci. 113, 1–34 (2013).

    CAS  Article  Google Scholar 

  2. 2

    Rodriguez, E.A. et al. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci. 42, 111–129 (2017).

    CAS  Article  Google Scholar 

  3. 3

    Shu, X. et al. Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324, 804–807 (2009).

    Article  Google Scholar 

  4. 4

    Shcherbakova, D.M. & Verkhusha, V.V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat. Methods 10, 751–754 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

    CAS  Article  Google Scholar 

  6. 6

    Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Choi, H.M.T. et al. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208–1212 (2010).

    CAS  Article  Google Scholar 

  8. 8

    Tanenbaum, M.E., Gilbert, L.A., Qi, L.S., Weissman, J.S. & Vale, R.D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

    CAS  Article  Google Scholar 

  9. 9

    Connally, R.E. & Piper, J.A. Time-gated luminescence microscopy. Ann. NY Acad. Sci. 1130, 106–116 (2008).

    CAS  Article  Google Scholar 

  10. 10

    Jin, D. et al. How to build a time-gated luminescence microscope. Curr. Protoc. Cytom. 67, 2.22 (2014).

    Google Scholar 

  11. 11

    Beverloo, H.B., van Schadewijk, A., van Gelderen-Boele, S. & Tanke, H.J. Inorganic phosphors as new luminescent labels for immunocytochemistry and time-resolved microscopy. Cytometry 11, 784–792 (1990).

    CAS  Article  Google Scholar 

  12. 12

    Marriott, G., Clegg, R.M., Arndt-Jovin, D.J. & Jovin, T.M. Time resolved imaging microscopy: phosphorescence and delayed fluorescence imaging. Biophys. J. 60, 1374–1387 (1991).

    CAS  Article  Google Scholar 

  13. 13

    Seveus, L. et al. Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization. Cytometry 13, 329–338 (1992).

    CAS  Article  Google Scholar 

  14. 14

    Marriott, G., Heidecker, M., Diamandis, E.P. & Yan-Marriott, Y. Time-resolved delayed luminescence image microscopy using an europium ion chelate complex. Biophys. J. 67, 957–965 (1994).

    CAS  Article  Google Scholar 

  15. 15

    Moore, E.G., Jocher, C.J., Xu, J., Werner, E.J. & Raymond, K.N. An octadentate luminescent Eu(III) 1,2-HOPO chelate with potent aqueous stability. Inorg. Chem. 46, 5468–5470 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Montgomery, C.P., Murray, B.S., New, E.J., Pal, R. & Parker, D. Cell-penetrating metal complex optical probes: targeted and responsive systems based on lanthanide luminescence. Acc. Chem. Res. 42, 925–937 (2009).

    CAS  Article  Google Scholar 

  17. 17

    Bünzli, J.-C.G. Lanthanide luminescence for biomedical analyses and imaging. Chem. Rev. 110, 2729–2755 (2010).

    Article  Google Scholar 

  18. 18

    Xu, J. et al. Octadentate cages of Tb(III) 2-hydroxyisophthalamides: a new standard for luminescent lanthanide labels. J. Am. Chem. Soc. 133, 19900–19910 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Heffern, M.C., Matosziuk, L.M. & Meade, T.J. Lanthanide probes for bioresponsive imaging. Chem. Rev. 114, 4496–4539 (2014).

    CAS  Article  Google Scholar 

  20. 20

    Dickson, E.F., Pollak, A. & Diamandis, E.P. Ultrasensitive bioanalytical assays using time-resolved fluorescence detection. Pharmacol. Ther. 66, 207–235 (1995).

    CAS  Article  Google Scholar 

  21. 21

    Hagan, A.K. & Zuchner, T. Lanthanide-based time-resolved luminescence immunoassays. Anal. Bioanal. Chem. 400, 2847–2864 (2011).

    CAS  Article  Google Scholar 

  22. 22

    Emami-Nemini, A. et al. Time-resolved fluorescence ligand binding for G protein-coupled receptors. Nat. Protoc. 8, 1307–1320 (2013).

    Article  Google Scholar 

  23. 23

    Connally, R., Jin, D. & Piper, J. High intensity solid-state UV source for time-gated luminescence microscopy. Cytometry A 69, 1020–1027 (2006).

    Article  Google Scholar 

  24. 24

    Gahlaut, N. & Miller, L.W. Time-resolved microscopy for imaging lanthanide luminescence in living cells. Cytometry A 77, 1113–1125 (2010).

    Article  Google Scholar 

  25. 25

    Rajendran, M. & Miller, L.W. Evaluating the performance of time-gated live-cell microscopy with lanthanide probes. Biophys. J. 109, 240–248 (2015).

    CAS  Article  Google Scholar 

  26. 26

    Moore, E.G., Samuel, A.P. & Raymond, K.N. From antenna to assay: lessons learned in lanthanide luminescence. Acc. Chem. Res. 42, 542–552 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Bünzli, J.C. & Piguet, C. Taking advantage of luminescent lanthanide ions. Chem. Soc. Rev. 34, 1048–1077 (2005).

    Article  Google Scholar 

  28. 28

    Armelao, L. et al. Design of luminescent lanthanide complexes: from molecules to highly efficient photo-emitting materials. Coord. Chem. Rev. 254, 487–505 (2010).

    CAS  Article  Google Scholar 

  29. 29

    Mathis, G. & Bazin, H. in Lanthanide Luminescence 47–88 (Springer, 2011).

  30. 30

    Byegård, J., Skarnemark, G. & Skålberg, M. The stability of some metal edta, dtpa and dota complexes: application as tracers in groundwater studies. J. Radioanal. Nucl. Chem. 241, 281–290 (1999).

    Article  Google Scholar 

  31. 31

    Firsching, F.H. & Brune, S.N. Solubility products of the trivalent rare-earth phosphates. J. Chem. Eng. Data 36, 93–95 (1991).

    CAS  Article  Google Scholar 

  32. 32

    Chen, J. & Selvin, P.R. Synthesis of 7-amino-4-trifluoromethyl-2-(1H)-quinolinone and its use as an antenna molecule for luminescent europium polyaminocarboxylates chelates. J. Photochem. Photobiol. Chem. 135, 27–32 (2000).

    CAS  Article  Google Scholar 

  33. 33

    Nishioka, T. et al. New luminescent europium(III) chelates for DNA labeling. Inorg. Chem. 45, 4088–4096 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Hanaoka, K., Kikuchi, K., Kobayashi, S. & Nagano, T. Time-resolved long-lived luminescence imaging method employing luminescent lanthanide probes with a new microscopy system. J. Am. Chem. Soc. 129, 13502–13509 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Thomas, D.D., Carlsen, W.F. & Stryer, L. Fluorescence energy transfer in the rapid-diffusion limit. Proc. Natl. Acad. Sci. USA 75, 5746–5750 (1978).

    CAS  Article  Google Scholar 

  36. 36

    Rajapakse, H.E. et al. Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells. Proc. Natl. Acad. Sci. USA 107, 13582–13587 (2010).

    CAS  Article  Google Scholar 

  37. 37

    Yam, V.W. & Wong, K.M. Luminescent metal complexes of d6, d8 and d10 transition metal centres. Chem. Commun. (Camb.) 47, 11579–11592 (2011).

    CAS  Article  Google Scholar 

  38. 38

    Thorp-Greenwood, F.L., Balasingham, R.G. & Coogan, M.P. Organometallic complexes of transition metals in luminescent cell imaging applications. J. Organomet. Chem. 714, 12–21 (2012).

    CAS  Article  Google Scholar 

  39. 39

    Botchway, S.W. et al. Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes. Proc. Natl. Acad. Sci. USA 105, 16071–16076 (2008).

    CAS  Article  Google Scholar 

  40. 40

    de Haas, R.R. et al. Phosphorescent platinum/palladium coproporphyrins for time-resolved luminescence microscopy. J. Histochem. Cytochem. 47, 183–196 (1999).

    CAS  Article  Google Scholar 

  41. 41

    Selvin, P.R. Lanthanide-based resonance energy transfer. IEEE J. Sel. Top. Quantum Electron. 2, 1077–1087 (1996).

    CAS  Article  Google Scholar 

  42. 42

    Kubota, T. et al. Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET. Proc. Natl. Acad. Sci. USA 114, E1857–E1865 (2017).

    CAS  Article  Google Scholar 

  43. 43

    Norris, K.P., Seeds, W.E. & Wilkins, M.H.F. Reflecting microscopes with spherical mirrors. J. Opt. Soc. Am. 41, 111–119 (1951).

    Article  Google Scholar 

  44. 44

    Miyata, S., Yanagawa, S. & Noma, M. Reflecting microscope objectives with nonspherical mirrors. J. Opt. Soc. Am. 42, 431–432 (1952).

    Article  Google Scholar 

  45. 45

    Witlin, B. Darkfield illuminators in microscopy. Science 102, 41–42 (1945).

    CAS  Article  Google Scholar 

  46. 46

    Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    CAS  Article  Google Scholar 

  47. 47

    Lu, Y. et al. Tunable lifetime multiplexing using luminescent nanocrystals. Nat. Photonics 8, 32–36 (2014).

    CAS  Article  Google Scholar 

  48. 48

    Chen, J.Y. & Selvin, P.R. Lifetime- and color-tailored fluorophores in the micro- to millisecond time regime. J. Am. Chem. Soc. 122, 657–660 (2000).

    CAS  Article  Google Scholar 

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This paper is dedicated to the memory of M. Buchin, whose technical expertise was invaluable for this project. We also thank D. Callard and J. Stepkowski (Stanford Photonics) for their assistance with our ICCD camera, C. Limouse for discussions about optical design and alignment, and D. Fitzpatrick and G. Gatmaitan (IOS Optics) for the design and fabrication of TiO2-coated coverglasses. This work was supported by a Samsung Scholarship (U.C.), a Stanford School of Medicine Dean's Fellowship (P.C.), the National Institutes of Health (DP1 HD075622 to J.K.C. and U01 HL099997 to P.B.H.), the National Science Foundation (CHE-1344038 to J.K.C.), and a Stanford ChEM-H Institute Seed Grant (J.K.C. and P.B.H.).

Author information




K.S.K. and J.K.C. built the time-resolved LED epifluorescence microscope; P.B.H. conceived the LRET-enhanced imaging by tuning lanthanide-lumiphore lifetimes; U.C. and P.B.H. conceived, designed, and built the QSL transreflected-illumination system; U.C., D.P.R., P.C., K.S.K., J.K.C., and P.B.H. designed the experiments; U.C. and P.C. performed the imaging experiments; U.C., D.P.R., P.C., K.S.K., J.K.C., and P.B.H. analyzed data; U.C., J.K.C., and P.B.H. wrote the paper.

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Correspondence to James K Chen or Pehr B Harbury.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–15 (PDF 3216 kb)

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Cho, U., Riordan, D., Ciepla, P. et al. Ultrasensitive optical imaging with lanthanide lumiphores. Nat Chem Biol 14, 15–21 (2018).

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