Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution

Abstract

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

Figure 1. Comparison of HIM and FE-SEM imaging in Arabidopsis thaliana.

(a) Low and (c) high magnification series of uncoated and critical point dried Arabidopsis thaliana sepal cuticle structures in the FE-SEM at low voltage (<1 kV) using an Everhart-Thornley (E-T) detector. At 22 kX magnification, charging artifacts become visible in the form of streaks or bands (white arrowheads) that go horizontally across the image. (b) Low and (d) high magnification series of the sepal cuticle structure in the HIM. Throughout the magnification range, no charging or imaging artifacts are apparent and cuticle structures are still visible at 163 kX. Scale Bars: (a,b) 10 μm (c,d) 200 nm.

Figure 2. Comparison of HIM and FE-SEM imaging of mitotic HeLa cells.

(a) Low magnification of uncoated critical point dried HeLa cells grown on glass coverslips in metaphase in both the FE-SEM at low voltage (<1 kV) with an In-Lens (I-L) detector (left panel) and HIM (right panel). Whilst the HIM exhibits high contrast and depth of field, the FE-SEM shows signs of charging in the form of saturated areas and dark and light banding on the glass coverslip. (b) At ~38 kX (3 μm field of view), the “ghosting” effect of some filopodia becomes apparent in the HIM (right panel, black arrowheads), while the FE-SEM exhibits a large amount of edge effects (left panel, black arrowheads). (c) High magnification of the same dividing cells shows no sign of loss of resolution in the HIM compared to the FE-SEM as depicted by the presence of cell membrane textures. Scale Bars: (a) 5 μm (b) 500 nm (c) 100 nm.

Figure 3. Single filopodia attachment point of a HeLa cell as illustrated using the HIM.

(a) A single cell was chosen that had minimal contact with neighboring cells and visible attachment points to the glass substrate. (b,c) The magnification was increased on a single adhesion point to a final magnification of (d) ~285 kX (400 nm field of view) while maintaining high enough contrast to depict an attachment width of ~5 nm. Scale Bars: (a) 5 μm (b) 1 μm (c,d) 50 nm.

Figure 4. Comparison of platinum coated and uncoated sample preparations critical point dried BoFeN1 bacteria in both the HIM and FE-SEM at low and high magnifications.

(a) Low and high magnifications of platinum coated (using an Everhart-Thornley (E-T) detector) and (b) uncoated bacterium (using an In-Lens (I-L) detector) in the FE-SEM reveal Fe(III) platelet structures that were on the order of tens of nanometers in size. Although the structures appeared much more flat in the uncoated sample, resolution was limited due to the necessity to use low-voltage imaging. (c) Low and high magnifications of platinum coated and (d) uncoated bacterium in the HIM reveal the coating induced artifact of globular structures and artificial textures on the platelets. The uncoated platelets in the HIM were for the first time revealed to be relatively flat with relatively sharp-tipped cellular attachments. Scale bars: 250 nm.

Figure 5. HIM imaging of Pristionchus pacificus.

(a) Native imaging of the nematode revealed the interior mouth cavity and tooth structures were obscured by a sheath-like structure. (b) neon milling of the outer sheath in the HIM revealed the primary tooth and DEGO structure of the nematode with minimal thermal damage to the cutting surface. (c) The removal of the outer sheath was performed in a very controlled manner with a final dose of 30 nC/μm³. (d) The delicate nature of the neon milling was demonstrated by the patterning of a small horizontal line using a dose of 0.3 nC/μm³. Despite being strong enough to penetrate the outer cuticle of the nematode, minimal ablation and thermal damage is visible on the surrounding area. Scale Bars: (a–c) 5 μm (d) 1 μm.