Figure 4. Multi-modal 3D cryogenic imaging by focused ion beam-scanning electron microscopy (FIB-SEM) volume imaging and cryo-electron tomography (cryo-ET).

Green modules represent tasks performed with SerialFIB; orange, with 3D Correlation Toolbox (3DCT). Gray modules represent tasks performed externally. (A) Focused ion beam (FIB) image of a Sum159 breast cancer cell prior to milling. Yellow rectangle indicates the volume for FIB-SEM volume data acquisition. (B) FIB image of the cell after FIB-SEM volume imaging. Yellow arrowheads indicate the defined position for lamella preparation. White arrowheads indicate micro-expansion joints prepared after volume imaging. (C) FIB image of the final lamella. (D) Representative slice of the raw FIB-SEM volume data and (E) postprocessed data, overlaid with manual segmentations of lipid droplets (dark blue) and the nucleus (cyan). (F) Slice through a tomogram acquired from lamella in (C). Inset shows the ribosome subtomogram average from data collected on lamellae prepared by this workflow (two tomograms; 3380 subtomograms; 24 Å resolution). (G) SEM view of two HeLa cells overlaid with a fluorescence light microscopy (FLM) maximum intensity projection of lipid droplet (red), mitochondria (cyan), and fiducial bead (yellow) fluorescence signals, determined by correlation in 3D Correlation Toolbox (3DCT). (H) Representative slice through the postprocessed FIB-SEM volume overlaid with a 200 nm virtual slice through the affine-transformed 3D-registered fluorescence volumes. White arrowhead points to a pair of lipid droplets, which are displayed at higher magnification in the inset.

Figure 4—figure supplement 1. Serial focused ion beam-scanning electron microscopy (FIB-SEM) volume imaging of C. reinhardtii and subsequent image processing adapted from Spehner et al., 2020.

(A) Raw data. Arrowheads indicate curtaining artifacts from ion beam milling. (B) De-striped image using wavelet decomposition to eighth level, Coiflets family 3 and Gaussian blurring (sigma = 6) of the vertical component. (C) Local charge adjusted by image subtraction of a Gaussian blurred (sigma = 35) and subsequently three times eroded image from the image in (B). (D) Image after median filtering (3.0 pixels) and local contrast enhancement using CLAHE (slope 3.0). (E–G) Zoom into regions indicated in (D), showing a Golgi stack (E, rectangle), thylakoid membranes (F), the nucleolus (E and G, dashed circles), and nuclear pore complexes in the nuclear envelope (G, arrowheads).

Figure 4—figure supplement 2. Point-based affine 3D registration between focused ion beam-scanning electron microscopy (FIB-SEM) and fluorescence volumes.

Registration in 3D Correlation Toolbox (3DCT) based on the unweighted centroids of lipid droplets and beads manually segmented in the FIB-SEM data, and centers of their fluorescence signal from Gaussian fitting in the corresponding fluorescence light microscopy (FLM) volume (n = 133). Lipid droplet and bead coordinates were pooled and used equally for the registration. (A) Fitted parameters for transformation of the FLM volume. Euler rotation angles are given in zxz convention. Shear factor s_yz represents shearing in y along the z axis. (B) 2D orthogonal projections of the FIB-SEM and transformed fluorescence coordinates. A gray line connects matching points between the two volumes. The root-mean-square-deviation of the fit was 386 nm, with residuals ranging from 29 nm to 882 nm.

Figure 4—video 1. Cryo-focused ion beam-scanning electron microscopy (cryo-FIB-SEM) volume of a Sum159 cell depicting raw and postprocessed data with segmentations of lipid droplets (blue) and the nucleus (cyan).

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Figure 4—video 2. Tomographic volume of a Sum159 breast cancer cell related to Figure 4F depicting the cytosol with two vault structures and a vesicle (V).

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Figure 4—video 3. Focused ion beam-scanning electron microscopy (FIB-SEM) volume of HeLa cells in Figure 4H, superposed with lipid droplet and bead segmentations and fluorescence volumes transformed.

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