Fig 1.
RGB composite images of the trace level element distribution of Ca, Zn and Fe (represented by the red, green and blue color channel respectively) of two single, high pressure frozen and cryosubstituted human neutrophils (white blood cells) before and after stimulation with phorbol myristate acetate (PMA), inducing the formation of so-called Neutrophil Extracellular Traps (or NETs).
NETs are newly discovered structures which are believed to act as a defense mechanism against microbes via chelating proteins, removing crucial trace elements from the pathogen. As all intensities in the trichromatic maps are normalized to a single upper value, increases in color brightness also represent increases in concentration. A clear increase in cellular Ca concentration is revealed via appearance of a yellow nucleus and reddish cytoplasm. Intracellular Fe and Fe/Ca-rich structures are also emerging after 2 h stimulation with PMA, visible as blue and pinkish hot-spots. The absent green color in the PMA-stimulated neutrophil indicates the strong association of Zn to Ca and/or Fe. Results were obtained using synchrotron radiation based X-ray fluorescence at the ID22NI beamline (European Synchrotron Radiation Facility, Grenoble) operating at a spatial resolution of 50 nm.
Fig 2.
RGB composite element maps of all quantitatively assessed neutrophils throughout PMA-stimulation. The red, green and blue color channels represent the intensities of the Ca, Zn and Fe distribution, respectively.
Upper row contains RGB composite element maps of neutrophils from control culture from donor A, middle and bottom row contain RGB composite element maps of neutrophils from donor B stimulated for 1–2 h with phorbol myristate acetate (PMA), respectively. All element intensities within the RGB composite element maps are normalized to diode current, dead time and dwell time per point (generally 300 ms). Maximum intensity of Ca, Zn and Fe (normalized) counts measured for the neutrophils from control culture were set as upper threshold values for the other RGB composite element maps. Within each RGB composite element map, the borders of nucleus and cytoplasm are indicated by a full and dashed white line, respectively. From each nucleus/cytoplasm area, an XRF sum spectrum was generated and used for quantification. Neutrophil nomenclature used in the quantitative analysis is provided within each RGB composite element map.
Fig 3.
a-b: Bar graphs with element weight fractions within entire neutrophils throughout PMA stimulation for the elements P, S, K, Cl, K, Ca, Mn, Fe, Co (upper graph, Fig 3a) and for Ni, Cu, Zn, Se, Br, Sr and Pb (lower graph, Fig 3b).
Neutrophils from control culture are indicated in green, 1 h PMA-stimulated neutrophils in blue and 2 h PMA-stimulated neutrophils in red. Weight fractions are expressed in %, ppm or ppb in a (different) logarithmic scale. Weight fraction values are normalized to the Compton intensity of neutrophil “0h_donorA_ cell1”. Error bars are based upon Poisson counting statistics and the certified uncertainty values of NIST SRM1577C “Bovine liver”.
Fig 4.
a-d: Bar graphs with element weight fractions within neutrophil nuclei (left column, Fig 4a-b) and cytoplasms (right column, Fig 4c-d) throughout PMA stimulation for P, S, K, Cl, K, Ca, Mn, Fe, Co (upper row) and for Ni, Cu, Zn, Se, Br, Sr and Pb (lower row).
Neutrophils from control culture are indicated in green, 1 h PMA-stimulated neutrophils in blue and 2 h PMA-stimulated neutrophils in red. Weight fractions are expressed in %, ppm or ppb in a (different) logarithmic scale. Weight fraction values are normalized to the Compton intensity of neutrophil “0h_donorA_ cell1”. Error bars are based upon Poisson counting statistics and the certified uncertainty values of NIST SRM1577C “Bovine liver”.
Table 1.
Mean weight fraction of P, S, Cl, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Br, Sr and Pb within entire neutrophils, their nuclei and cytoplasms from control culture and 1–2 h PMA-exposed culture.
Table 2.
Statistical data-analysis upon mean weight fractions of entire neutrophils (a), their nuclei (b) and cytoplasms (c) throughout PMA stimulation.