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.

Comparison of Preparation Methods of Bacterial Cell-Mineral Aggregates for SEM Imaging and Analysis Using the Model System of Acidovorax sp. BoFeN1

“…This is only briefly discussed here for the sake of completeness; it is more thoroughly described by Joens et al (2013). The fact that minerals appear rounded in Pt-coated samples can be attributed to preferential Pt accumulations at exposed structures such as edges and tips, likely caused by charge-effects during the sputter-coating process.…”

“…Another important feature is the fact that an electron flood gun can be used for charge compensation, which allows for the analysis of nonconductive samples. In this way, uncoated samples may be imaged without charging artifacts (Joens et al 2013). Resin embeddingSamples of a 3-week-old BoFeN1 culture were embedded in Spurr's resin and GMA resin.Samples were taken under anoxic conditions and washed twice with Fe-free culture medium, which included centrifugation, in order to remove potentially remaining Fe 2+ ions.…”

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

“…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). Additionally, there is no need for metal coating of the samples, due to the unique charge compensation mechanism in HIM (Joy, 2013, Hlawacek et al, 2014.…”

“…The latter is very different from SEM and important to reach the estimated resolution of 0.35 nm. 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 combination of such characteristics has made HIM an instrument reaching ultra-high resolution (down to 0.35 nm) and a promising tool for the analysis of biological surfaces, as elegantly shown by Rice et al (2013), who characterized the surface of kidney cells using this microscope, and by Vanden Berg-Foels et al (2012), who analyzed bone tissue. Other examples on the potential and reliability of HIM were given by Bazou et al (2011) analysis of colon cancer cells and, more recently, by Joens et al (2013) for the study of plants, HeLa cells, bacteria and nematodes as well as Pȃunescu et al (2014) describing aspects of the male reproductive tract of rats. Novel characteristics on the cytoskeleton of Giardia lamblia have also been reported by observing detergent extracted trophozoites (Gadelha et al, 2015).…”