aIn vivo micro X-ray fluorescence (micro-XRF) on plants is aimed at mapping spatial ion accumulation, eg. in plant leaves to minimize artifacts arising from sample preparation and to be able to detect changes occurring under environmental stress. To obtain reliable results, a spectrometer and sample setup that maintain the plant in good physiological condition are necessary. We have built a micro-XRF spectrometer, in which analyses are performed in air, thereby allowing for in vivo micro-XRF measurements in plants. Mapping is performed with full spectrum acquisition in each pixel and is automated and controlled by computer software developed for this micro-XRF unit. Limits of detection were found to be comparable to those found previously at second-generation synchrotron facilities using a freestanding film reference from 0.04 ng/mm 2 for Fe to 0.20 ng/mm 2 for Pb. The spot size was determined to be less than 14 μm at Rh Kα line (20.214 keV). Relative sensitivities were determined for elements from S to Pb using the freestanding film reference and a 13-element standard dried droplet deposition. For in vivo measurements, a dedicated sample holder was developed by 3-D printing. Here, we present the in vivo elemental analysis capabilities of our micro-XRF spectrometer by mapping elements in Arabidopsis thaliana plants and by assessing their physiological state before and after scanning.