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.