FIGURE 2.

CLEM using IRF samples. (A) Array tomography. 100 nm thick serial sections of a human skin biopsy, high-pressure frozen, freeze-substituted with uranyl acetate, osmium tetroxide and Safranin O, and embedded in HM20 were mounted on ITO-coated glass coverslips. This approach allows multiple on-section labeling to complement IRF. The top subset was imaged by fluorescence LM without further staining, using the Safranin O signal introduced during FS, the middle subset shows on-section staining with Toluidine, and the subset of sections was stained with Dapi. Following LM, all sections were imaged by SEM at 2 kV using in-lens SE detection. Combination of all images yields a volume that comprises multiple levels of information, ideal for in-depth characterization of complex biological structures. Volume size (x-y-z): LM stack: 321.0 × 221.5 × 5.1 µm³, SEM stack: 226.4 × 170.0 × 5.1 µm³. (B) 3D CLEM of root nodule from mung bean (Vigna radiata), colonized with nitrogen-fixing bacteria (B. japonicum). 200 µm thick sections were high-pressure frozen after degassing in 1-hexadecene, followed by FS and embedding in HM20. The FS-medium contained uranyl acetate, osmium tetroxide and Acridine orange for fluorescent labeling of the bacteria. En bloc CLSM was performed to identify a ROI, which was subsequently targeted by FIB-SEM. The CLSM and FIB-SEM volumes were merged using the Amira software package. The imaging plane of FIB-SEM is perpendicular to that of CLSM. Volume size (x-y-z): CLSM 57.2 × 57.2 × 9.0 µm³, FIB-SEM 21.8 × 6.2 × 15.9 µm³. Figure adapted from Lucas et al. (2012) with permission of Elsevier Ltd.