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Uniformly dispersed luminescent probes with a high brightness and high resolution are desired in bio imaging fields. Here, ~100 nm sized and well-dispersed spheres of RE³⁺ doped α-NaYF₄ (rare earth (RE) = Eu, Tb, Ce, Er, and Tm) have been facile synthesized through hydrothermal processing in the absence of a template, followed by a proper annealing. The processing window of the cubic structured spheres is wide, because the hydrothermal products are independent of the processing conditions, including reaction time and temperature. The original morphology and crystal structure can be well retained with a calcination temperature up to 600 °C. However, calcination gives rise to a reduction of particle sizes, as a result of the crystallite growth and densification. Under ultraviolet radiation, α-NaYF₄:RE³⁺ spheres show characteristic f-f emissions of RE³⁺ (RE = Eu, Tb, Ce, Er, and Tm), and exhibit orange red, green, ultraviolet (UV), blue green, and blue emissions, respectively. Mainly because of the near-infrared emission at ~697 nm (⁵D₀→⁷F₄ transitions of Eu³⁺), the successful imaging of macrophages was achieved by NH₂-NaYF₄:Eu³⁺ probes, indicating their excellent imaging capacity for cells in vitro.