Rare-earth-doped nanoparticles are one of the emerging probes for bioimaging due to their visible-to-near-infrared (NIR) upconversion emission via sequential single-photon absorption at NIR wavelengths. The NIR-excited upconversion property and high photostability make this probe appealing for deep tissue imaging. So far, upconversion nanoparticles include ytterbium ions (Yb3 + ) codoped with other rare earth ions, such as erbium (Er3 + ) and thulium (Tm3 + ). In these types of upconversion nanoparticles, through energy transfer from Yb3 + excited with continuous wave light at a wavelength of 980 nm, upconversion emission of the other rare earth dopants is induced. We have found that the use of the excitation of Er3 + in the 1550-nm wavelength region allows us to perform deep tissue imaging with reduced degradation of spatial resolution. In this excitation–emission process, three and four photons of 1550-nm light are sequentially absorbed, and Er3 + emits photons in the 550- and 660-nm wavelength regions. We demonstrate that, compared with the case using 980-nm wavelength excitation, the use of 1550-nm light enables us to moderate degradation of spatial resolution in deep tissue imaging due to the lower light scattering coefficient compared with 980-nm light. We also demonstrate that live cell imaging is feasible with this 1550 nm excitation.
We describe rare-earth-doped nanophosphors (RE-NPs) for biological imaging using cathodoluminescence (CL) microscopy based on scanning transmission electron microscopy (STEM). We report the first demonstration of multicolor CL nanobioimaging using STEM with nanophosphors. The CL spectra of the synthesized nanophosphors (Y2O3:Eu, Y2O3:Tb) were sufficiently narrow to be distinguished. From CL images of RE-NPs on an elastic carbon-coated copper grid, the spatial resolution was beyond the diffraction limit of light. Y2O3:Tb and Y2O3:Eu RE-NPs showed a remarkable resistance against electron beam exposure even at high acceleration voltage (80 kV) and retained a CL intensity of more than 97% compared with the initial intensity for 1 min. In biological CL imaging with STEM, heavy-metal-stained cell sections containing the RE-NPs were prepared, and both the CL images of RE-NPs and cellular structures, such as mitochondria, were clearly observed from STEM images with high contrast. The cellular CL imaging using RE-NPs also had high spatial resolution even though heavy-metal-stained cells are normally regarded as highly scattering media. Moreover, since the RE-NPs exhibit photoluminescence (PL) excited by UV light, they are useful for multimodal correlative imaging using CL and PL.
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