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[Preprint]. 2023 Jul 12:2023.07.11.548578. [Version 1] doi: 10.1101/2023.07.11.548578

Figure 2. 3D cryo-multi-modal correlation workflow in an iFLM-FIB-SEM system.

Figure 2.

iFLM guided FIB-milling procedure via the single pair ROI-Marker 3D correlation in a typical cryo-FIB milling session. (1) The registration of SEM (A) and iFLM (B) of a HeLa cell with lipid droplets (green), and mitochondria (red), and nucleus (blue) to show the XY location information of the ROI (single lipid droplet, yellow arrowhead). (2) The transformation of 3D Z positioning is achieved via the Z plane difference between the ROI (yellow arrowhead, D, Z slice of 22) and the marker (1 μm bead, red box, em 605 nm, E, Z slice of 17). (3) The transformation of lateral information and Z-position from SEM/iFLM to the FIB via the milling angle on the targeted cell to set the initial location for rough milling. (4) A second round of registration and transformation between the same ROI-Marker in XYZ is performed at the end of the rough milling (thickness of 3 μm). The automated milling process was not interrupted to produce ~200 nm lamellae. The entire procedure is integrated in the AutoTEM automated milling workflow (AutoTEM 2.4.2). The screenshots of FIB correlative localization in AutoTEM (F and I). Scale bars of 50 μm in A and B, 20 μm in C (2 μm in the zoom-in inset), and 10 μm in D, E, G, H.