Correlative cellular cryo-ET workflow for robust quantitative analysis of membrane ultrastructures between distinct mitochondrial network morphologies and treatment conditions. (A) MEFmtGFP cells were cultured on transmission EM grids (black mesh circle) and treated with vehicle or Tg (500 nM) for 8 h prior to vitrification via plunge freezing in ethane/propane mixture. (B) Vitrified MEFmtGFP cells were imaged using cryo-fluorescence microscopy (cryo-FM). (C) Cryo-FM facilitated the identification of single cells with distinct elongated or fragmented bulk mitochondrial network morphologies. (D) These cells were targeted for cryo-FIB milling to generate thin sections (lamellae). (E) Lamella were imaged using standard cryo-ET imaging parameters to generate tilt series that were further reconstructed to generate 3D cryo-tomograms. (F) Voxel segmentations labeling distinct cellular membranes were generated using a semiautomated approach (Martinez-Sanchez et al., 2014) prior to conversion to point clouds. Next, a normal-oriented vector is estimated for each point (Cignoni et al., 2008). Normal-oriented point clouds were used to generate surface meshes using a novel application of a screened Poisson reconstruction method followed by masking to the original voxel segmentation (Kazhdan and Hoppe, 2013; Cignoni et al., 2008). (G) Surface meshes model the implicit 3D ultrastructure of distinct organellar membranes. Surface coloring: IMM, purple; OMM, blue; ER, teal. (H) Implicit geometries encoded within surface meshes are then used to perform 3D morphometrics to quantify several parameters that define membrane ultrastructure across mitochondria from distinct morphological and treatment conditions.
