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The developing nanotechnology at single-molecule level promotes the study of single-particle performance.For up-conversion colloidal nanoparticles, the microscopic crystalline structure and the surface bonding dependent luminescence is critical for their promising applications.Here we show the site-selective spectroscopic splitting of Er3+ levels using single wavelength excitation at room temperature, which present the evidence that the up-conversion luminescence between the surface sites and intemal sites in the oleic-acid capped β-NaYF4∶Yb3+-Er3+ single nanoparticles are different from emission profile to transition mechanism.Moreover, we show a direct observation of the bottleneck effect in up-conversion efficiency from the β-NaYF4∶Yb3+-Er3+ colloidal nanoparticles in high excitation condition by single-particle spectroscopy, which is totally different from the enhanced UC phenomenon in β-NaYF4∶Yb3+-Tm3+ system.