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. 2021 Feb;281(2):138-156.
doi: 10.1111/jmi.12951. Epub 2020 Aug 24.

An introduction to cryo-FIB-SEM cross-sectioning of frozen, hydrated Life Science samples

M F Hayles  1 , D A M DE Winter  2
Affiliations

An introduction to cryo-FIB-SEM cross-sectioning of frozen, hydrated Life Science samples

M F Hayles et al. J Microsc. 2021 Feb.

Abstract

The introduction of cryo-techniques to the focused ion-beam scanning electron microscope (FIB-SEM) has brought new opportunities to study frozen, hydrated samples from the field of Life Sciences. Cryo-techniques have long been employed in electron microscopy. Thin electron transparent sections are produced by cryo-ultramicrotomy for observation in a cryo-transmission electron microscope (TEM). Cryo-TEM is presently reaching the imaging of macromolecular structures. In parallel, cryo-fractured surfaces from bulk materials have been investigated by cryo-SEM. Both cryo-TEM and cryo-SEM have provided a wealth of information, despite being 2D techniques. Cryo-TEM tomography does provide 3D information, but the thickness of the volume has a maximum of 200-300 nm, which limits the 3D information within the context of specific structures. FIB-milling enables imaging additional planes by creating cross-sections (e.g. cross-sectioning or site-specific X-sectioning) perpendicular to the cryo-fracture surface, thus adding a third imaging dimension to the cryo-SEM. This paper discusses how to produce suitable cryo-FIB-SEM cross-section results from frozen, hydrated Life Science samples with emphasis on 'common knowledge' and reoccurring observations. LAY DESCRIPTION: Life Sciences studies life down to the smallest details. Visualising the smallest details requires electron microscopy, which utilises high-vacuum chambers. One method to maintain the integrity of Life Sciences samples under vacuum conditions is freezing. Frozen samples can remain in a suspended state. As a result, research can be carried out without having to change the chemistry or internal physical structure of the samples. Two types of electron microscopes equipped with cryo-sample handling facilities are used to investigate samples: The scanning electron microscope (SEM) which investigates surfaces and the transmission electron microscope (TEM) which investigates thin electron transparent sections (called lamellae). A third method of investigation combines a SEM with a focused ion beam (FIB) to form a cryo-FIB-SEM, which is the basis of this paper. The electron beam images the cryo-sample surface while the ion beam mills into the surface to expose the interior of the sample. The latter is called cross-sectioning and the result provides a way of investigating the 3rd dimension of the sample. This paper looks at the making of cross-sections in this manner originating from knowledge and experience gained with this technique over many years. This information is meant for newcomers, and experienced researchers in cryo-microscopy alike.

Keywords: Cross-section; FIB-SEM; cryo-FIB-SEM; cryo-fracture; serial-sectioning; site-specific X-sectioning; vitrification.

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Figures

Fig. 1
Fig. 1
Cryo‐FIB‐SEM setup: (A) The FIB‐SEM as installed at Utrecht University. In the middle the microscope itself and to the right additional hardware related to the cryo‐transfer system. Most of the results shown in this paper are obtained with this setup. (B) Schematic representation of the position of the sample with respect to the FIB and SEM. Both beams view the same location on the sample when the eucentric position is used. Working at the eucentric position is beneficial, as the SEM can check the progress of ongoing FIB‐milling actions.
Fig. 2
Fig. 2
Cryo‐transfer hardware: (A) The cryo‐stage with inlet/outlet tubes for the cold Nitrogen gas and electrical connections for the temperature read‐out and heating element. (B) The Quorum PP2000T prep chamber with freeze fracturing, sublimation, sputter coating facilities and enabling a cold, vacuum transfer into the FIB‐SEM chamber. (C) The transfer rod is used to interface between the freezing unit (external), the prep chamber and the Microscope chamber. (D) The cryo‐holder/sledge comes in a variety of types for different applications and can contain one or more samples. Customizing these parts is easy and may be necessary to suit the experiment.
Fig. 3
Fig. 3
Commercially available carriers: (A) 10 mm aluminium block with fracture recesses for LN2 only. (B) Rivets for plunge freezing/fracture in suitable cryogen. (C) Planchets used the same as rivets. (D) Grids are usually associated with cryo‐TEM but can be used for cross‐sections if sample concentration is higher than regular TEM suspension. (E) Microstub and sapphire disc for HPF where thin layers are useful. (F), (G) Gold membrane carriers (1.5 mm diameter, 100 or 200 µm thick), two for a mid‐volume fracture, one + sapphire disc for surface fracture. Bases for (E) to (G) are custom made from 10 mm half‐height aluminium stubs. (All drawings not to scale.)
Fig. 4
Fig. 4
Cryo‐fracture: This frozen yeast culture demonstrates how temperature affects fracture. (A) Elastic fracture due to too high temperature ( –150 °C, showing fracture has moved over and around cells and not through. (B) Normal fracture for SEM, some though cells and others showing fractured P2 and P3 membranes. (C) Glassy fracture, straight through most cells. Not ideal for SEM but perfect for cryo‐FIB‐SEM cross‐sectioning. Scale bar: 5 µm.
Fig. 5
Fig. 5
Creating a cryo‐fracture surface with two rivets. (A) The flat surfaces of both rivets are roughened with sandpaper to increase the contact surface between the rivets and the sample. (B) The rivet‐ends are closed with TissueTekTM to prevent leakage. A droplet of sample is placed on the lower rivet. (C) The second rivet is carefully placed on top. (D) The ensemble is frozen by lowering it vertically into LN2 slush or liquid ethane/propane. (E) A precooled knife knocks off the top rivet when in the transfer station, creating a fracture surface. (F) The fracture surface is sputter coated with a metal to prevent electrical charging. It is now ready for transfer into the cryo‐FIB‐SEM.
Fig. 6
Fig. 6
Contamination types: (A), (B) Examples of LN2 born ice crystals deposited on the surface. (C), (D) Ice crystals condensed on the surface from air exposure. (E), (F) Temperature differential contamination “orange peel” caused by a warm surface in the close vicinity of the sample. (G) Debris from the fracture process. (H) Redeposition from the ion beam milling process. The redeposition is recognisable from the sides of the cross‐section which become increasingly ‘bulging’ in front of the cross‐section during prolonged milling. Scale bars: (A) 50 µm, (B) 10 µm, (C) 5 µm, (D) 50 µm, (E) 2 µm, (F) 2 µm, (G) 100 µm and (H) 20 µm.
Fig. 7
Fig. 7
Sublimation: Tobacco leaf structure. (A) After FIB‐milling showing low contrast, (B) after sublimation, removing minimal water to give contrast. (C) Yeast cell after sublimation displaying Mitosis. Scale bars: (A) and (B) 30 µm and (C) 1 µm.
Fig. 8
Fig. 8
Curtaining and protection: (A) Surface roughness causing severe curtaining. (B) Cryo‐sample showing Au/Pd sputter coating (bright line), followed by contaminating ice (dark line) from transfer, influencing the protection layer by insufficient release of gas on planerising. In turn the gas voids create curtaining. (C) An uneven surface (Mildew spore pod), covered by protective deposition, to improve and reduce the chance of curtaining. Scale bar for (A) 10 µm, (B) 2 µm and (C) 10 µm.
Fig. 9
Fig. 9
Contrast: (A) After FIB‐milling. (B) Creating contrast by sublimation. (C) Surface charging at 3 kV (more contrast, more charge effect). (D) Subsurface charging at 5 kV (less visual charge, more depth effect). The latter two images show the influence of different SEM beam energies and how the use and reproducibility can be a challenge. Scale bars: (A), (B) 3 µm, (C) and (D) 5 µm.
Fig. 10
Fig. 10
Localised beam damage: (A) cryo‐FIB‐SEM E‐beam damage after fracture and before milling of P2 inner membrane (Yeast cell) seen as swelling and splitting. Invaginations and particle arrays are visible. (B) TEM image of a cryo‐FIB‐SEM prepared lamella of yeast cells, demonstrating ‘bubbling’ of cellular component due to excessive exposure to the TEM E‐beam. The FIB lamella has a thickness of 300 nm and although damaged, typical structures of S. cerevisiae are seen; multivesicular bodies (MVB), mitochondria (M), lipid granules (LG) and cortical endoplasmic reticulum (ER) are clearly visible. (C) Swelling of the cell walls due to over E‐beam scanning. Scale bars: (A) 500 nm, (B) 500 nm, (C) 5 µm.
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