Ca2Si5N8:Eu2+,Tm3+ presents outstanding long lasting luminescence at about 610 nm. However, to be useful for in vivo optical imaging, persistent luminescence materials should possess high optical performance combined with sizes in the nanoscale. With this aim, we investigated two different techniques for the preparation of nanoparticles from Ca2Si5N8:Eu2+,Tm3+ bulk powder. First, nanoparticles were successfully prepared with the pulsed laser ablation method in liquid (abbreviated as PLAL). Secondly, nanoparticles obtained by selective sedimentation from the bulk compound resulted in satisfactory yield and allowed to perform the first real-time in vivo imaging with Ca2Si5N8:Eu2+,Tm3+ host. Finally the influence of surface functionalization on the biodistribution of the probe after systemic injection is discussed.