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. 2021 Jun 10;24(7):102714. doi: 10.1016/j.isci.2021.102714

Figure 2.

Figure 2

3D correlation using lipid droplets as fiducial markers

(A) Overlay of a low-magnification cryo-FM and cryo-SEM (InLens SE/SE2 mixed detection) showing the same area on the EM grid. Cells of interest are identified in the cryo-SEM using an asymmetric mark at the center of the grid (white square and magnified on right) and other landmarks on the grid. Cells were labeled with BODIPY493/503 (green), which stains LDs. LDs are subsequently used as internal fiducial markers for CLEM.

(B) Overlay of a higher magnification cryo-FM and cryo-SEM showing a cell of interest.

(C) Maximum intensity projection of a focus series showing BODIPY distribution in a cell of interest. Arrows (green and yellow) correspond to LDs shown in the micrographs in (I). Scale bar: 5 μm.

(D) Quantification of the number of LDs per cell among potential cells for CLEM. The average number is 37 ± 22 LDs/cell (N = 63, median = 29 LDs/cell).

(E) Two representative cryo-FIB-SEM micrographs of the correlated cell; LDs (yellow circles) are clearly visible. Voxel size: 10 × 10 × 10 nm. Scale bar: 1 μm.

(F) Average z-projection of the error prediction map per image after affine image transformation using 17 LDs. The error prediction ranges from 370 (purple) to 880 nm (white).

(G) The transformed FM micrograph (gray) rendered as a volume including spheres (red) indicating the space in which the actual fiducial is with 95% confidence.

(H) Transformed grid slice of xy-plane based on the affine transformation showing the slight stretch of the image in the y and z directions (scaling of 27% and 39%, respectively, z deformation is not shown). This is probably caused by artifact induced from tilt correction and line averaging (on the y axis) and minute differences in the slice thickness and stack alignment (on the z axis).

(I) Two representative micrographs showing the overlay of the transformed FM on top of the FIB-SEM dataset (magnified ×1.5 from E). The affine transformation allowed for correlation of both LDs in the high-LDs-density area (green) as well as the low-LDs areas (orange).