A method for multi-parametric x-ray fluorescence imaging with maximized detection sensitivity and minimized radiation dose for a biological/living sample (10) containing a first marker substance comprises the steps of irradiation of the sample (10) with x-ray radiation (1), with x-ray fluorescence (2) of the first marker substance being excited, spatially resolved detection of the x-ray fluorescence (2) of the first marker substance, and determination of a distribution of the first marker substance in the sample (10) from the x-ray fluorescence (2) of the first marker substance, wherein the sample (10) contains at least one further marker substance which is excited to x-ray fluorescence (2) by the x-ray radiation (1), wherein fluorescence lines (3) of the first and the at least one further marker substances are different, at least one of the first and the at least one further marker substances is coupled with active ingredient molecules and/or ligand molecules provided for a specific interaction with the sample (10) or contained in cells, in order to be able to trace these, the detection comprises a spectrally resolved detection of the x-ray fluorescence (2) of the first and the at least one further marker substances, and additionally at least one distribution of the at least one further marker substance in the sample (10) is determined from the detected x-ray fluorescence (2) of the first and the at least one further marker substances. An imaging device (100) for multi-parametric x-ray fluorescence imaging and an optimized selection method for a marker substance kit for introducing marker substances into a sample (10) are also described.
