HPF and cryo-FIB lift-out

D. melanogaster egg chambers were dissected from ovaries in Schneider’s medium and HPF (HPM010, Abrafluid) in the 100 µm cavity of gold-coated copper Type A carriers (Engineering office M. Wohlwend) using Schneider’s medium containing 20% Ficoll (70 kDa) as filling medium. HPF carriers were soaked in hexadecene and blotted on filter paper prior to freezing. Cryo-planing of approximately 40 µm of the surface was performed in a cryo-microtome (EM UC6/FC6 cryo-microtome, Leica Microsystems) using a 45° diamond trimming knife with a clearance angle of 6° (DiAtome). The sample was glued to a custom 3D-printed shuttle after the Leica design (Kuba et al., 2021) using cryo-glue (2:3 ethanol:isopropanol mixture) for cryo-confocal imaging with a Leica TCS SP8 equipped with Leica EM cryo-CLEM stage. Imaging was performed using a HC PL APO ×50/0.9 DRY objective in fluorescence mode using a 552 nm excitation laser at 17% total laser strength and 488 nm excitation at 6.8% total laser strength, corresponding to roughly 0.94 mW and 0.38 mW, respectively (based on measured laser intensity values). Detection channels were 650–751 for mCherry and 495–545 nm for autofluorescence using HyD detectors at a pinhole size of 2 Airy units. Z-stacks of 38 µm from the surface were collected using a step size of 1 µm. Fluorescence signal decreased significantly with imaging depth. Targeting of positions for automated trench milling was based on the surface topology of the sample produced by the cryo-planing. Positions were chosen based on registration with cryo-FLM data in 3DCT, which allowed the identification of regions within the egg chamber.

For lift-out experiments, an Aquilos dual-beam FIB-SEM microscope (Thermo Fisher Scientific) was equipped with a Kleindiek MM3A-EM micromanipulator and a cryo-gripper head (Kleindiek Nanotechnik) cooled by copper wires attached to the microscope anti-contaminator. Standard 45° pre-tilt shuttles were used for accommodating both HPF carrier and Auto-Grid clipped receiver grid on an Aquilos stage that was modified by removing ~2 mm of material of the stage using a file to allow the cryo-gripper to reach the sample surface. OmniProbe 4-post Cu half-grids were used as receiver grids (Plano EM). After specimen loading, platinum GIS deposition was performed for 10 s at 27°C with the GIS needle distance at a stage working distance of 10.6 mm. Samples were sputter coated with platinum in the chamber (10 mA, 15 s). For all trench milling steps and SerialFIB session setup, drift suppression using the SEM was applied to compensate positive charges brought in by FIB milling. The stage was rotated by 180° relative to loading position and tilted to 7° to orient the sample surface perpendicular to the FIB. Trench milling was performed with ‘regular cross-section patterns’ at 30 kV, 1 nA creating a C-shape using SerialFIB (Figure 5). The milling included two patterns of 40 µm × 15 µm symmetrically arranged around a 20 µm block. Together with a third pattern, 10 µm × 40 µm was generated at an offset of 15 µm to the left and perpendicular to the previous two, yielding a 20 µm × 20 µm block for lift-out. Milling time per position was 30 min. In total, five positions were prepared, resulting in a milling preparation time of 2.5 hr. Undercuts were performed manually by tilting the stage to 43° and employing ‘cleaning cross-section’ patterns of 1 µm height, 22 µm width, and 15 µm depth at ~7 µm distance from the sample surface. Lift-out was performed with the microgripper oriented parallel to the FIB angle and perpendicular to the sample surface. The produced sample chunks were approached by the gripper. After contact of both tweezer arms with the sample, the chunk was milled loose from the bulk material with ‘regular cross-section patterns’ at 1 nA with a pattern of 20 µm in length, 1 µm in width until extraction from the bulk was possible. Subsequently, the microgripper was raised until it was ~400 µm above the sample surface. The stage was moved down by ~15 mm into safe distance from the gripper and subsequently moved to the receiver grid position while keeping the Z-height locked. Then, the stage was lifted to the appropriate Z-height of the receiver grid. The microgripper was moved to the post of the TEM half-grid until achieving contact of the sample. ‘Regular cross-section’ patterns at height of 2 µm and width of 6 µm were used to attach the sample to the post by re-deposition of the post material onto the sample. Subsequently, the gripper was opened, releasing the chunk that has been welded to the pin, and moved to a safe position. This is achieved by lifting the gripper until it is 400 µm above the sample surface and then moving the stage down by 15 mm, making enough room to safely move it to the park position. This procedure can be performed for all four pins. Finally, deposition of platinum GIS for 3 × 30 s in 3 min intervals at a stage working distance of 9 mm was used to strengthen the sample attachment to the pin. Lamella production was done manually with sequentially reduced FIB currents of 3 nA to 5 µm lamella thickness, 1 nA to 4 µm, 0.5 nA to 3 µm, 0.3 nA to 2 µm, and 0.1 nA to 1 µm using ‘regular cross-sections’ patterns. Subsequent fine milling was performed using ‘regular patterns’ at 0.1 nA to 600 nm and 50 pA to 200 nm. The FIB divergence was compensated by over- and under-tilts of 1° (Schaffer et al., 2017). The lamella was milled until contrast started fading in SEM imaging at 3 kV, 13 pA.