**2.2. Measurement setup**

The LA-ICP-MS measurements were performed using a quadrupole-based ICP-MS (XSeries 2, Thermo Fisher Scientific, Bremen, Germany) without operating the collision cell. The Nd:YAG laser ablation unit (New Wave UP 213, New Wave, Fremont, CA, USA) was coupled directly to the ICP-MS. Measurement parameters such as RF power and gas flow were optimized for each sample with a daily performance test. Typical LA-ICP-MS measurement parameters that we used in our experiments for simultaneous detection of 21–29 different isotopes were: RF power, 1400–1600 W; carrier gas flow, 0.85–1.05 l/min; dwell time per isotope, 0.02–0.3 s; maximal energy at sample surface, > 3 mJ; fluence at sample surface, >30 J/cm2 ; scan speed, 60–90 μm/s; spot size, 30 μm; and *y*-distance between the center of the lines, 60 μm, respectively. To keep the integration time of the mass spectrometer as low as possible and for receiving a high spatial resolution in the future image, only isotopes of interest were selected. The dwell time was adjusted to the sensitivity of the detector for each selected isotope.

For the measurement, the glass slide bearing the sample tissue section was positioned on the *xyz*-stage in the laser ablation chamber and moved into the focus plain (*z*-direction) of the laser beam. Within the focus correction option, the *z*-position was adjusted to the starting and ending point of every line to compensate minor variations in inclination and tilt of the sample. The ablation was performed line by line with a continuous motion in the *x*-direction and a discontinuous motion in the *y*-direction. At the end of each line, the laser was interrupted and moved back to the zero *x*-position with *y*-offset of 30 μm to the next line, while sample material was continuously transported to the mass spectrometer. The primary output data were a continuous list of ion intensities with no trigger signal indicating line shifts, because the mass spectrometer operated independently from the laser unit.

Before each measurement, the LA-ICP-MS was equilibrated to obtain constant conditions. Therefore, the ablation chamber was washed by constant carrier gas flow for about one hour to remove air entered during loading. As a function of equilibration and washing, *y*-drifts occurred in some data sets, but also during prolonged measurement runs, i.e., over 12 h. For calibration, matrix-matched standards were measured at the end of the same run. These were produced from homogenized tissue of a homologous organ spiked with different concentrations of a standard solution [11]. Sample and standard were present at separate slides positioned side by side into the chamber.

For image generation, the LA-ICP-MS raw data of individual measurements were copied into a standardized Excel summary file, whereupon the user-defined functions (UDF) of ELAI could be accessed. Once the image dimensions (line length) have been found, the absolute element concentrations of each data point were determined from its ion intensity and the slope of a six-point calibration curve. The software tool "ELAI" [8] that we have recently developed is open software offering free customizable semimanual image generation. The complete program, including detailed documentation for installation and disclosure of all program codes is freely available and can be downloaded from the supplementary material of [8].
