Imaging of immunogold labeling in cells and tissues by helium ion microscopy

Int J Mol Med. 2018 Jul;42(1):309-321. doi: 10.3892/ijmm.2018.3604. Epub 2018 Mar 30.

Abstract

Helium ion microscopy (HIM) scans samples with a fine ion beam exploiting the very short de Broglie wavelength of helium ions. Because the radiation induces only a small sample region to emit secondary electrons (SEs), very high resolution is expected. In order to explore the applications of SE-HIM in biology, COS7 kidney fibroblast cells and C2C12 myoblast cells cultured on a silicon (Si) nitride (SiN)/Si bilayer were dried and directly observed in high vacuum, without coating or staining. High contrast, high depth-of-field images were obtained revealing the nucleus, endoplasmic reticulum, cytoskeleton and putative mitochondria above a bright background from the support. Gold-tagged antibodies were employed to aid organelle identification. Signals from the gold tags were most clearly distinguishable by secondary electron (SE)-HIM when cells were grown on thin SiN film, and the minimum gap measured between gold particles showed the resolution to be 2 nm. Wheat germ agglutinin-gold labeling revealed clusters of gold particles ~50-200 nm in diameter on COS7 cells, which might represent assemblies of glycosylated proteins, suggesting the formation of membrane raft structures that include membrane proteins. SE-HIM also delivered high contrast images of unstained, uncoated, thin sections of Epon‑embedded mouse kidney tissues mounted on a SiN/Si bilayer, revealing the details of sub-tissues and cell organelles. A charge-coupled mechanism explaining the observed SE-HIM contrast is proposed. Ionoluminescence-HIM was also performed targeting zinc oxide particles on cells. In conclusion, the high depth-of-field, high-resolution imaging achieved using HIM may have applications in various fields, including soft materials.

MeSH terms

  • Actins / metabolism
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Colloids / chemistry
  • Endoplasmic Reticulum / metabolism
  • Fibroblasts / cytology*
  • Fibroblasts / metabolism
  • Fluorescence
  • Helium / chemistry*
  • Immunohistochemistry / methods*
  • Ions
  • Kidney / cytology*
  • Mice
  • Microscopy / methods*
  • Microtubules / metabolism
  • Mitochondria / metabolism
  • Myoblasts / cytology*
  • Myoblasts / metabolism
  • Polysaccharides / metabolism
  • Silicon / chemistry
  • Silicon Compounds / chemistry
  • Staining and Labeling*
  • Stress Fibers / metabolism
  • Tubulin / metabolism

Substances

  • Actins
  • Colloids
  • Ions
  • Polysaccharides
  • Silicon Compounds
  • Tubulin
  • Helium
  • silicon nitride
  • Silicon