Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography

Abstract

Vitreous freezing offers a way to study cells and tissue in a near-native state by cryo-transmission electron microscopy (cryo-TEM), which is important when structural information at the macromolecular level is required. Many cells - especially those in tissue - are too thick to study intact in the cryo-TEM. Cryo focused-ion-beam (cryo-FIB) milling is being used in a few laboratories to thin vitreously frozen specimens, thus avoiding the artifacts and difficulties of cryo-ultramicrotomy. However, the technique is challenging because of the need to avoid devitrification and frost accumulation during the entire process, from the initial step of freezing to the final step of loading the specimen into the cryo-TEM. We present a robust workflow that makes use of custom fixtures and devices that can be used for high-pressure-frozen bulk tissue samples as well as for samples frozen on TEM grids.

Keywords: Cryo-FIB; Cryo-SEM; Cryo-TEM; Cryo-tomography; Vitreous sections.

Figure 1

Intermediate Specimen Holder (ISH). Two types are shown. On the left in (A) is an ISH designed for holding a standard HPF carrier, which is cut down (nearly in half in this case, see Fig. 3). to expose the tissue. On the right in (A) is an ISH designed for a TEM grid. A. Top and bottom views: Channels on the bottom (arrowheads) fit into the TEM cryo-transfer holder (Fig. 8). The central hole (short arrow) accepts the locating pins in both the loading block (Fig. 2) and in the TEM cryo-transfer holder (Fig. 8). The side holes (long arrows) allow the ISH to be easily manipulated by forceps. B. Side views of both types of ISH (grid-type on the left and HPF-carrier type on the right): The long arrows show the holes where the loading-box retractable pin inserts to spread the jaws. The lower hole forms the hinge. Bars = 1 mm.

Figure 2

ISH loading block. A. The knob on the left spreads the jaws of the ISH by inserting the pin, shown by the arrow in (C). B. View of an ISH in the loading block. The countersunk holes in the loading block are for re-positioning the ISH, if necessary. C. Side view of the empty loading block showing the locating pin (arrowhead) and the spreading pin (arrow). Bars: A = 3 mm; B,C = 1 mm.

Figure 3

Pre-trimming geometry. A. An HPF carrier, held in an ISH. Nearly half of the carrier has already been trimmed away. The two arrows show the 0.7-mm-wide region (with the tissue in the center) to be further trimmed, as shown in (B) and (C). B. Tissue (*) in a 300-μm-wide slot of the aluminum HPF carrier, before final trimming. At this stage, a layer of aluminum is still beneath the tissue. C. Final trimming step. The edge of the tissue is trimmed to 20 μm thickness, supported by aluminum only at the sides. Bars: A = 1 mm; B,C = 100 μm.

Figure 4

Leica specimen block. A. Shown with the cover open. Note the thick, curved structures on both ends; these act as additional anti-contaminators, and they also restrict the flow of gas into the ends of the block. In the inset, two ISHs are shown mounted in the block, one with a TEM grid (arrow) and one with a partially-trimmed HPF carrier (arrowhead). B. Shown with the shutter closed. The shutter automatically opens when the block is released in the FIB/SEM coldstage, and it automatically closes when the block is retracted. Bars = 1 cm (3 mm in inset).

Figure 5

VCT-100 cryo-transfer system and vent gas arrangement. A. The shuttle (*) is attached to the loading box and the gate valve (arrowhead) is opened. The specimen block (Fig. 4) is placed under liquid nitrogen by the transfer rod inside the shuttle. This operation is done both when picking up the specimen block before FIB-milling and retrieving it afterwards. B. The specimen block (black arrow) is introduced into, or removed from, the loading box by means of a handling rod (white arrow). After transfer from the microtome chamber, or before transfer to the TEM cryo-holder workstation, the specimen block, attached to the rod, is stored in a 50-ml plastic tube (black arrowhead) of liquid nitrogen that is kept in a large Dewar. C. The shuttle (*) is shown attached to the MED 020 evaporator with the shuttle gate valve (arrowhead) and the dock gate valve both open. The shuttle is vented on the MED 020 coating unit, and the vent gas needs to be free of any water vapor. D. The shuttle (*) is attached to the FIB-SEM with both gate valves initially closed. E. A copper coil submerged in a Dewar of liquid nitrogen cools lightly pressurized (8-10 psi) nitrogen gas that is boiled off in a 50-liter Dewar (not shown). F. The anticontaminator in the VCT-100 shuttle is cooled with liquid nitrogen, which can be seen in the reservoir, indicated by the black arrow. Prior to venting, the lines are purged using the added purge valve, shown by the white arrow.

Figure 6

Types of frost, as deposited on Quantifoil test films with 2-μm-diameter holes. A. If there is water vapor in the vent gas, very small (ca. 50 nm) frost particles are seen. B. The frost is almost eliminated using the arrangement in Fig. 5. C. Larger frost particles may be deposited on the specimen while the specimen block is in the loading box, and can also be deposited while mounting the specimen in the ISH in the cryo-microtome chamber. Bars = 2 μm.

Figure 7

FIB-milling steps. A. An end view (“ion-beam image”) showing the full 20 μm tissue thickness (double arrowheads) at the far left and far right, milled in the center to about 60 μm deep and 10 μm thick (Step 1, 5 nA). B. Two regions are further milled to about 40 μm deep and 3 μm thick (Step 2, 1 nA). The shape of the trapezoid-shaped milling pattern is outlined at the left region. C. The right-side region from (B) was further milled to about 150-250 nm thick in four sub-areas (Step 3, 100 pA). The arrow indicates the direction of view in (D) and (E). D. An SEM side-view image--perpendicular to the view in (C)--of the final four TEM lamellae. E. A low-mag TEM view of the four lamellae shown in (D), each about 20 μm deep and 10 wide. The two lamellae on the left were damaged during transfer. Bars: A,B = 20 μm; C,D,E = 10 μm.

Figure 8

TEM cryo-transfer holder in its workstation. A. The FIB-SEM specimen block is opened under liquid nitrogen (but shown here dry), exposing an ISH to be transferred. Both TEM-grid (arrow) and HPF-carrier (arrowhead) samples are shown, but normally only one ISH is used at a time. B. The recess for the ISH is revealed with the cover (black arrow) open. The white arrow shows the locating pin for the ISH. The fastening screw is shown with arrowheads in (B) and (D). C. An ISH with a TEM grid is inserted into the recess, but the cover is still open. D. The cover is closed and secured with a captive screw (arrowhead). The window revealing the TEM grid is apparent. E. The cryo-transfer shutter closed, ready for transfer to the TEM. Bars: A = 1 cm; B-E = 5 mm.

Figure 9

Example of results -- FIB sections of vitreously frozen muscle tissue (from toadfish). A. Section approximately 150 nm thick, recorded at 400 keV with zero-loss energy filtering and 2 e^-/Ų incident dose. The sarcoplasmic reticulum vesicles (SR) and t-tubule cross-sections (TT) are surrounded by muscle fibers. Profiles of ryanodine receptors (RyR) are shown at the arrows; in this view, two RyRs are arranged next to each other on both sides of the TT. In this “side view” of the RyRs, the RyR's transmembrane domain (illustrated in the inset as the structure projecting to the right of the RyR's main mass), is inserted in the SR membrane so that only the larger, cytoplasmic region appears in the gap between SR and TT. B. Electron diffraction pattern indicating vitreous ice. C. Cryo-SEM image of the very thin lamella (*) corresponding to the TEM image in (A). D. Projection image of a 300-nm-thick section, recorded as above, showing a row of five triad junctions. E. Projection image of a ∼100-nm-thick slice recorded as in (A). F, G. 1-nm-thick tomographic slices of the triad junctions in (E), left and right respectively; pixel size 1 nm, total dose for the tilt series 100 e^-/Ų. Bars: A,D = 100 nm; E,F,G = 50 nm.