Whole-Cell Imaging at Nanometer Resolutions Using Fast and Slow Focused Helium Ions

Abstract

Observations of the interior structure of cells and subcellular organelles are important steps in unraveling organelle functions. Microscopy using helium ions can play a major role in both surface and subcellular imaging because it can provide subnanometer resolutions at the cell surface for slow helium ions, and fast helium ions can penetrate cells without a significant loss of resolution. Slow (e.g., 10-50 keV) helium ion beams can now be focused to subnanometer dimensions (∼0.25 nm), and keV helium ion microscopy can be used to image the surfaces of cells at high resolutions. Because of the ease of neutralizing the sample charge using a flood electron beam, surface charging effects are minimal and therefore cell surfaces can be imaged without the need for a conducting metallic coating. Fast (MeV) helium ions maintain a straight path as they pass through a cell. Along the ion trajectory, the helium ion undergoes multiple electron collisions, and for each collision a small amount of energy is lost to the scattered electron. By measuring the total energy loss of each MeV helium ion as it passes through the cell, we can construct an energy-loss image that is representative of the mass distribution of the cell. This work paves the way to use ions for whole-cell investigations at nanometer resolutions through structural, elemental (via nuclear elastic backscattering), and fluorescence (via ion induced fluorescence) imaging.

Tipping solutions: emerging 3D nano-fabrication/ -imaging technologies

“…an electron column is added to image the FIB fabrication region using the standard SEM technique. High-resolution imaging is also possible using a focused He-ion beam [79,80].…”

“…Given that the scattering of helium ions is very sensitive to the mass of the target atoms (particularly for high-atomicnumber (ions), incubating heavy ion nanoparticles (NPs) inside the sample is needed. Chen et al reported on the 3D whole-cell imaging of gold nanoparticle in cell biological system through the combination of STIM for imaging the cell structure [79], forward STIM for lateral image of gold NPs, and Rutherford backscattering spectrometry (RBS) for depth profiles of gold NPs. The spatial resolutions of STIM and RBS are ~25−30 nm and 200−300 nm, respectively.…”

A review of critical factors for assessing the dermal absorption of metal oxide nanoparticles from sunscreens applied to humans, and a research strategy to address current deficiencies

“…Nor can they distinguish endogenous metal from metal ions in, or sourced from, the metal oxide particles. However, the best resolutions achieved with nuclear microscopy are below 50 nm (Chen et al 2011). Such level of resolution would possibly enable individual particle detection and visualization on the basis of density contrast, which is equivalent to TEM.…”

New views of the Toxoplasma gondii parasitophorous vacuole as revealed by Helium Ion Microscopy (HIM)

“…The gas field ionization source has ultra-high brightness, and a very small beam defining aperture may be used, minimizing spherical and chromatic aberrations and resulting in higher depth of field and a estimated resolution of 0.35 nm (Joy, 2013;Hlawacek et al, 2014;Joens et al, 2013). Besides that, when the surface of the sample is bombarded by He + , the penetration of the beam is smaller, when compared to an electron beam accelerated to 30 kV, improving the resolution (Chen et al, 2011). The secondary electron yield is much higher than in SEM, giving to the images a better signal-to-noise ratio (Inai et al, 2007;Joens et al, 2013).…”