This work reports on synchrotron X-ray fluorescence (SR-XRF) imaging of vitrified islets of Langerhans in two-dimensional and computed tomography (CT) mode and serves as a proof-of-principle for the preparation and SR-XRF imaging of cryoloops in a cryogenic environment. Selection of suited cryoloops and cryoprotectant solution enabled vitrification of the islets. The SR-XRF experimental setup was adapted with a cryogenic gas stream, enabling analysis of cryofrozen islets and a dual silicon drift detector (SDD) configuration, providing an increased solid angle for XRF collection. Element distributions of K, Ca, Fe and Zn within single, cryofrozen islets could be obtained with 500 nm spatial resolution using X-ray compound refractive lenses (CRLs) with an adaptive phase plate. Titanium, present as a trace element in the cryoloops, was used as a marker signal to efficiently define the XRF scan area. Preliminary 2D cryo-analysis of wild type ( n = 1) and Mt1-Mt2 -knock-out ( n = 1) islets under low/high glucose conditions did not reveal changes in the distribution and concentration of Ca, Fe and Zn. Cryotomography XRF scans indicated aspherical islets within the cryoloop, likely due to capillary effects in the cryoloop before plunge freezing. The presence of diffracting ice crystals hampered CT reconstruction, indicating the need for further development of a routine workflow ensuring perfect vitrification. Collectively, this novel multidisciplinary approach proved to be valid and can be used for future studies to assess elemental compositions of islets of Langerhans or other cell clusters/tissues under different pathophysiological conditions. This work reports on trace level chemical imaging of vitrified islets of Langerhans in 2D/CT mode using synchrotron X-ray fluorescence (SR-XRF). The newly developed method can be used for other biological samples that can be captured in a cryoloop.