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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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.).
The authors declare no competing financial interests.
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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). https://doi.org/10.1038/nchembio.2513
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