Sample preparation for X-ray Fluorescence Microscopy
C. elegans were prepared for X-ray Fluorescence Microscopy (XFM) using previously described protocols [1, 2].
Developmentally synchronised adult C. elegans were removed from Nematode Growth Agar (NGM) plates via a standard flattened platinum pick, washed four times in excess S-basal (0.1 M NaCl; 0.05 M KHPO4 at pH 6.0), and then briefly in ice-cold 18 MΩ resistant de-ionized H2O (Millipore).
Two final washes in ice-cold CH3COONH4 (1.5 % w/v) was used to both reduce dissolved salts.
Approximately 5-20 adults were then gently transferred onto 0.5 um-thick silicon nitride (Si3N4) window (Silson) in a minimum of buffer using a glass Pasteur pipette.
The excess buffer was wicked away using fine paper wicks (MiTeGen).
As required an eyelash was used to reposition the samples in the centre of the Si3N4 window.
The windows were then dipped into in liquid nitrogen (N2)-chilled liquid propane using a KF-80 plunge freezer (Leica Microsystems), and placed in a heavy platter (100 mm dia x 100 mm chromed brass) that had been prechilled with liquid N2 to ensure the samples remained froze.
The samples were then lyophilised overnight at -40 °C and then stored under low vacuum to prevent rehydration until required.
References
1. James, S.A., et al., Direct in vivo imaging of essential bioinorganics in Caenorhabditis elegans. Metallomics, 2013. 5(6): p. 627-35.
2. Hare, D.J., et al., High-resolution complementary chemical imaging of bio-elements in Caenorhabditis elegans. Metallomics, 2016. 8: p. 156-160.