Cryo-Stem Tomography Provides Morphological and Chemical Characterization of Precipitated Calcium-Phosphate Clusters Sequestered in Mitochondria of Intact Vitrified Fibroblasts

Abstract

We recently demonstrated that cryo-scanning transmission electron tomography (CSTET) provides tomographic reconstructions of vitrified cells with superior information transfer at high tilts and for thicker specimens than defocus phase contrast (Wolf et al., 2014). In cryoSTEM, there are no image-forming lenses after the electron beam passes through the sample; detection is incoherent and inelastically scattered electrons provide usable contrast information. By obviating the need for zero-loss energy filtration, the STEM modality provides efficient use of electron dose, thereby minimizing specimen damage.Here we demonstrate the use of CSTET for obtaining highly detailed 3D architectures of organelles and macromolecular complexes in unstained, unfixed, and unsectioned cultured fibroblasts while simultaneously collecting analytical information from high-angle, incoherently scattered electrons. As a case in point, cryoSTEM tomograms revealed characteristic patterns of dense deposits sequestered in mitochondria. Energy-dispersive X-ray (EDX) spectroscopy of these deposits revealed calcium and phosphorus. Once the elemental identification was made, the STEM scattering signal could be interpreted quantitatively as a three-dimensional map of mitochondrial calcium deposition. This approach can be extended to identify and map other concentrations of elements in the cell heavier than the pervasive carbon, nitrogen, and oxygen, as we demonstrated for phosphorus in bacterial cells (Wolf et al., 2015). This study provides an example of how imaging with sensitivity to atomic number in whole cells will provide a new dimension in structural cell biology by correlating elemental composition to organelle morphology.ReferencesWolf SG, Houben L and Elbaum M, “Cryo-scanning transmission electron tomography of vitrified cells”, Nature Methods 11(4):423-428 (2014).Wolf SG, Rez P and Elbaum M, “Phosphorus detection in vitrified bacteria by cryo- STEM annular dark field analysis”, J. Microscopy http://dx.doi.org/10.1111/jmi.12289.