New insights on mitochondrial heterogeneity observed in prepared mitochondrial samples following a method for freeze-fracture and scanning electron microscopy

Mitochondria are dynamic intracellular organelles with diverse roles in tissue- and cell type-specific processes, extending beyond bioenergetics. In keeping with this array of functions, mitochondria are described as heterogeneous both between and within tissue types based on multiple parameters, including a broad spectrum of morphological features, and new research points toward a need for the evaluation of mitochondria as isolated organelles. Although transmission electron microscopy (TEM) is commonly used for the evaluation of mitochondria in tissues and renders mitochondria structures in ultra-thin sections in two-dimensions, additional information regarding complex features within these organelles can be ascertained using scanning electron microscopy (SEM), which allows for analysis of phenotypic differences in three-dimensions. One challenge in producing mitochondrial images for evaluation of ultrastructure using SEM has been the ability to reliably visualize important intramitochondrial features, the inner membrane and cristae structures, on a large-scale (e.g. multiple mitochondria) within a sample preparation, as mitochondria are enclosed within a double membrane. This can be overcome using a 'freeze-fracture' technique in which mitochondrial preparations are snap-frozen followed by application of intense pressure to break open the organelles, revealing the intact components within. Previously published reports using freeze-fracture strategies for mitochondrial SEM have demonstrated feasibility in whole tissue specimens, but a detailed methodology for SEM analysis on isolated mitochondrial fractions has not been reported. By combining previously reported tissue freeze-fracture strategies, along with utilizing the depth of field created by SEM, herein we present a complete method reliant on the freeze-fracture of mitochondria' fractions prepared by differential centrifugation to produce a comprehensive and direct evaluation of three-dimensional mitochondrial ultrastructure by SEM. Image analysis of internal mitochondrial features demonstrates heterogeneity in mitochondrial ultrastructure from a single sample preparation.