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In modern biology and medicine, research methods based on the use of luminescent nanomarkers are actively developed. The main problem of using luminescent agents in biological environment is the isolation of the useful signal of marker from the background caused by autoluminescence of the biological medium. This problem can be solved by using nanocomplexes based on lanthanides that have intense up-conversion luminescence when excited in the near-IR range. A characteristic feature of these markers is that their luminescent response strongly depends on the mode of excitation of luminescence. In this case, important parameter is not only the intensity of pumping, but also the type of excitation - continuous or pulsed radiation. The case of excitation by short powerful laser pulses is of particular interest. In this study, suspensions of new synthesized NaYF4:Yb/Tm nanocomplexes in dimethylsulfoxide were studied. In order to excite up-conversion luminescence, a laser system based on a pulsed Nd:YAG laser and optical parametric oscillator was used, which made it possible to achieve the pumping intensity of 300 MW/cm2 at the wavelength 980 nm. The most important factor determining the features of the appearance of up-conversion luminescence of NaYF4:Yb/Tm nanocomplexes under pulsed excitation is the mechanism of its formation. In the studied complexes, it is realized by transferring the energy of exciting radiation from ytterbium (”sensitizer“) to thulium ("activator"), therefore, the most important parameters are the pumping intensity and the ratio of the concentrations of activator ions and sensitizer ions. In this study, two series of samples were prepared: in the first series, the concentration of the sensitizer changed, while the concentration of the activator remained constant, in the second series, the concentration of the activator changed at constant concentration of the sensitizer. During the experiment, the dependences of the intensity of the most pronounced thulium luminescence bands on the intensity of the pumping radiation when excited by high-intensity pulsed laser radiation were obtained and analyzed. The obtained results made it possible to clarify the models of the mechanisms of up-conversion luminescence of NaYF4:Yb/Tm complexes and determine the optimal ratio of the concentrations of sensitizer and activator.