Anatomical Study of Cells and Tissues using Helium Ion Microscopy

Helium Ion Microscopy (HeIM) has advantages for the study of cells and tissues, not available with other ultra-microscopy techniques like Scanning Electron Microscopy (SEM). Imaging without an electroconductive coating of the sample, reveals more details of biological structures (an insulating material) compared to conductivity-coated SEM, making HeIM an ideal component for correlative microscopy analysis, combining nanoscale imaging with optical spectroscopy. The lack of coating allows samples to be re-imaged easily with fluorescence or optical microscopy in a reversible process, allowing multiple data sets to be acquired for the same sample. We show how a minimal sample preparation (often a simple formalin fixation) permits the analysis of a larger number of specimens in the same unit time compared to other techniques that demand multiple steps. We report our specific experience in employing HeIM based correlative microscopy on cells (Vero E6, Schwann cells and oligodendrocytes) and tissue (brain, nerve, retina, kidney). For instance, HeIM shows clearly the morphological network of microfilaments (filopodia, lamellipodia or tunneling nanotubes), after fluorescence microscopy highlighted the presence of fibrillar actin (filament forming proteins). In the case of tissue imaging, the absence of coating provided surface details of fine structure on nerve tissue devoid of shrinkage artifacts in myelinated fibers.