To compare SEM to HIM, a set of biological samples were chosen where the unique aspects of the HIM system could provide advantages in their imaging. (i) Arabidopsis thaliana, the most favored model system for plant biology to study exterior nanoscale morphology (ii) HeLa cells, as an adherent cell type to study membrane surface details and filopodia, (iii) BoFeN1 iron-oxidizing bacteria to showcase the effects of drying and heavy metal coating on surface morphology and (iv) Pristionchus pacificus predator nematodes to illustrate the depth of field of the HIM system and its precision milling abilities.

Imaging parameters were chosen to provide a rational comparison between the two technologies. Since the overarching goal of the study was to investigate uncoated samples as well as to achieve high-resolution images at high magnifications, the FE-SEM was operated at a low voltage (0.7–1.0 kV for uncoated samples and 3 kV for coated samples) with an aperture size of 30 μm. Both In-Lens (I-L) and Everhart-Thornley (E-T) detectors were used to image the samples as only secondary electrons were studied in the HIM. Alternatively, the HIM was operated at an optimal imaging voltage of 30 kV, an aperture size of 5 μm and a beam blanker current of 0.5 pA. An Everhart-Thornley (E-T) detector was used to image the samples. In both instances, working distance and tilt were slightly varied between images and samples to produce optimal imaging conditions of electron detection, depth of field and charge compensation.

Care was taken during sample preparation to ensure that any artifacts were minimal. To reduce drying artifacts and to make the samples vacuum stable, proper fixation and critical point drying (CPD) was necessary. It was found that small plants and nematodes could be fixed with ice-cold ethanol and then directly critical point dried. Cells, however, were more sensitive to the CPD process and required the use of robust fixatives such as glutaraldehyde to maintain intact membrane structures. In addition all samples were kept in a desiccator or under vacuum at all times to minimize artifacts caused by rehydration of the tissues from native humidity.

Arabidopsis thaliana

As a model plant, Arabidopsis was chosen as a stable and well-studied organism to compare the effects of imaging at ever increasing magnification ranges between the FE-SEM and HIM. Since the samples were critical point dried and uncoated, they were prone to charging and thus low voltage (<1 kV) imaging in the FE-SEM was required. When compared to the HIM, images in the SEM were similar at lower magnifications in both contrast, depth of field and lack of charging (Figure 1a and b panels). However, despite low-voltage imaging in the FE-SEM, the sample exhibited charging effects starting at ~20 kX magnification (3.4 μm field of view), resulting in motion shown by streaks or bands within the image (Figure 1c, white arrowheads). In addition, at ~38 kX (2.1 μm field of view) fine textures on the sample surface and minute ridges on the cuticle could no longer be discerned. By 163 kX (478 nm field of view), the cuticle could no longer be clearly resolved in the FE-SEM as a result of either heavy charging or beam-induced thermal drift due to the very small probe size necessary to achieve such a magnification (Figure 1c far right panel). In contrast, charging in the HIM was completely eradicated via the use of the microscope's charge-compensation flood gun. As a result, higher magnifications up to 163 kX could be easily achieved without any form of charging, thermal drift and most importantly beam-induced damage, thus revealing fine surface structures, such as the defects in the wax on a single ridge of the plant's cuticle (Figure 1d panels).

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. Full size image

HeLa cells

As a model cell line used in many cell biology applications, HeLa cells were chosen to demonstrate the effects of imaging fine structures such as filopodia and cell adhesion points in both HIM and SEM. The comparison of two cells in mitosis at low magnification (Figure 2a) clearly shows the efficient charge neutralization ability of the HIM over the FE-SEM. The intensity-saturated regions of the cell as well as the appearance of dark bands on the glass coverslip indicated the areas of charging in the FE-SEM. As the magnification was increased to 38 kX (3 μm field of view), two separate effects can be seen within each microscope. In the HIM, filopodia had a tendency to appear “ghosted” or somewhat translucent when there were additional features in close z-proximity (Figure 2b, right panel, black arrowheads). As described previously, this is likely due to the deeper penetration of the helium ions that pass through thinner regions of the sample, generating not only SE1 electrons on the surface closest to the source, but also secondary electrons on the surfaces immediately behind them31. This is due to the ion beam not changing geometry over that distance. In the SEM, the filopodia exhibited edge effects, a common artifact of low-voltage imaging of fine or filamentous structures (Figure 2b, left panel, black arrowheads). As magnification was increased a third time to 163 kX (700 nm field of view) (Figure 2c), the filopodia were easily recognizable in both microscopes, however, the surface features and textures were indistinguishable in the FE-SEM (Figure 2c, left panel).

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. Full size image

In addition to the comparison of the HIM to the SEM, the maximum overall resolution of the HIM was demonstrated in Figure 3. A random HeLa cell was chosen (Figure 3a) and from there, a single filopodia attachment point was recorded at increasing magnifications (Figure 3b–d) until a maximum magnification of 285 kX, or a field of view of 400 nm was achieved. At this magnification, the sample contrast between the cell and the glass coverslip was still high enough to discern the size of the attachment point (~5 nm). Remarkably, repeated imaging of these small and highly delicate structures caused no discernable beam damage.

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. Full size image

Iron-oxidizing bacteria (BoFeN1)

The nitrate-reducing Acidovorax sp. strain BoFeN1, originally isolated from anoxic freshwater sediments of Lake Constance causes oxidation of Fe(II) either by an enzymatic process or by an abiotic reaction of Fe(II) with nitrite, which is an intermediate denitrification product32. It is known from numerous previous studies that, when grown in the presence of Fe(II), some cells tend to grow a crust of platelet-shaped Fe(III) minerals on the cell surface while others do not33.

The HIM images that were acquired at low magnification show three-dimensional cell mineral aggregates (Figure 4c and d, left panels). The images show a large depth of field in comparison to FE-SEM images that were acquired at low voltage at similar magnifications (Figure 4a and b, left panels). Low voltages were necessary in particular for imaging the uncoated samples by FE-SEM to avoid charging artifacts. The comparison of coated and uncoated samples by HIM at high resolution reveals two major artifacts caused by the sputter coating with platinum. Comparing the coated and non-coated critical point dried samples (Figure 4c and d) it appears that the crystallites attached to the cells end in rather sharp tips without coating, whereas mineral platelets on the cell surfaces of the platinum-coated samples appear to be relatively thick at the end and sometimes even show globule-like structures in the size-range of tens of nanometers (Figure 4c, right panel). A potential explanation for this change in mineral shape and structure might be a localized, preferential deposition of platinum due to charge-effects on the electrically isolating mineral structures. Additionally, the critical point dried samples show especially clean surfaces, whereas the surfaces of the minerals in plunge-frozen and freeze-dried samples appear to have a much smoother envelope in the case of the uncoated samples. We attribute this to a thin layer of extracellular polymeric substances (EPS) coating the whole aggregate. In contrast, we observed a relatively rough surface in the case of the platinum-coated samples (see Supplementary Fig. 1). This indicates that sputter coating with platinum introduces an artificial surface roughness.

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. Full size image

HIM imaging has, for the first time, allowed the identification of this coating artifact because of the higher spatial resolution in combination with the capability of analyzing non-conductive, uncoated samples. In addition, the existence of an EPS-envelope coating the minerals in plunge-frozen and freeze-dried samples could only be unambiguously shown in non-coated samples. Furthermore, sputter coating with platinum seemed only to affect the surface of the chemically fixed and critical point dried bacteria by creating a textured surface with network-like structures that created high secondary electron-signals (Figure 4c). We did not observe this effect when the cells were not coated with platinum (Figure 4d). We also did not observe these structures on plunge-frozen and freeze-dried samples, both with and without the heavy metal coating (see Supplementary Fig. 1). Since dehydration in solvents such as ethanol (a necessary step preceding critical point drying), can cause shrinkage in biological samples34, it is also possible that a similar effect could lead to a slightly textured cell surface that is potentially electrically insulating and thus, could favor localized deposition of platinum due to charge induced effects.

Predator nematode - pristionchus pacificus

Finally the predator nematode, Pristionchus pacificus35 served as an ultrastructural imaging challenge in the sense that the interior of the mouth cavity, including teeth morphology, had never been successfully imaged. A sister nematode, Parasitodiplogaster laevigata, a parasite of fig wasps has previously been shown via SEM imaging to have a protruding tooth structure and a visible Dorsal Esophogeal Gland Orifice (DEGO) located at the base of the protruding tooth36. However, in the initial HIM imaging of Pristionchus pacificus, it was revealed that a membranous sheath obscured the primary tooth structure (Figure 5a). This afforded an opportunity to employ another unique aspect of the HIM system – precision nano-machining using focused noble gas ions. By switching out the working gas of the HIM from helium to neon, it was possible to delicately remove the tip of the membranous sheath, while leaving the interior of the mouth cavity intact (Figure 5c). This reveals the interior mouth structure as well as the primary tooth morphology and DEGO shape and size (Figure 5b). The very precise milling ability of the neon beam was effectively demonstrated by cutting the exterior of the nematode with a final dose of 0.3 nC/μm3 (Figure 5d). This proved intense enough to cut through the outer skin of the worm without damaging any of the surrounding tissue. For deeper cuts (such as the one shown in Figure 5c), the final cutting dose was increased to 30 nC/μm3 to penetrate the entire depth of the worm and efficiently mill through some of the tougher, more filamentous portions of the sample such as the mechanosensory fibers around the nematode head.