Three-dimensional imaging of mitochondrial cristae complexity using cryo-soft X-ray tomography

Abstract

Mitochondria are dynamic organelles that change morphology to adapt to cellular energetic demands under both physiological and stress conditions. Cardiomyopathies and neuronal disorders are associated with structure-related dysfunction in mitochondria, but three-dimensional characterizations of the organelles are still lacking. In this study, we combined high-resolution imaging and 3D electron density information provided by cryo-soft X-ray tomography to characterize mitochondria cristae morphology isolated from murine. Using the linear attenuation coefficient, the mitochondria were identified (0.247 ± 0.04 µm−1) presenting average dimensions of 0.90 ± 0.20 µm in length and 0.63 ± 0.12 µm in width. The internal mitochondria structure was successfully identified by reaching up the limit of spatial resolution of 35 nm. The internal mitochondrial membranes invagination (cristae) complexity was calculated by the mitochondrial complexity index (MCI) providing quantitative and morphological information of mitochondria larger than 0.90 mm in length. The segmentation to visualize the cristae invaginations into the mitochondrial matrix was possible in mitochondria with MCI ≥ 7. Altogether, we demonstrated that the MCI is a valuable quantitative morphological parameter to evaluate cristae modelling and can be applied to compare healthy and disease state associated to mitochondria morphology.

Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells

“…By SXT we can resolve the internal structure of mitochondria, revealing the organization of cristae and detect areas of close apposition of vesicular organelles, resembling LE/LYSs. The complex mitochondrial ultrastructure, we observe by SXT in human fibroblasts is in accordance with recent X-ray and electron microscopy studies of mitochondria in muscle cells 39 , 40 . It supports the potential of these methods in delineating mitochondrial structure and morphology in human diseases.…”

“…To assess mitochondrial ultrastructure and resolve MCSs at the nanoscale, we employ X-ray microscopy. SXT provides three-dimensional information with isotropic resolution ≤ 50 nm, and since its contrast is based on linear absorption of X-rays, the extent of ‘darkness’ in reconstructed transmission images is a quantitative measure of X-ray absorption by cellular structures 38 , 39 . By SXT we can resolve the internal structure of mitochondria, revealing the organization of cristae and detect areas of close apposition of vesicular organelles, resembling LE/LYSs.…”

Role of Mitochondria in Physiology of Chondrocytes and Diseases of Osteoarthritis and Rheumatoid Arthritis

“…55,56 Enzyme complexes involved in electron transport in the respiratory chain are located on the IMM. 57 ATP is synthesized from adenosine diphosphate (ADP) via ATP synthase which is located in the end of the ETC. 58 During the process of OXPHOS, 4 ETC complexes (respiratory chain complexes I-IV) and complex V coordinate to generate ATP and provide energy for chondrocytes.…”

The use of soft X-ray tomography to explore mitochondrial structure and function

“… Require use of thin lamella sections of cells that may not accurately reflect cellular organization due to polarity and uneven distributions of cellular contents [ [58] , [59] , [60] ] Fluorescence Microscopy (confocal, super-resolution) 70–250 nm in a single cell Provides live cell dynamics and cellular rearrangements Requires specific labeling strategy limiting unbiased discovery [ [61] , [62] , [63] , [64] ] Soft X-Ray Tomography (cells cryo-fixed on a grid or in solution within capillary. Fluorescence correlative) Up to 35 nm in 10 μm diameter cell Produces unbiased and high-throughput 3D reconstructions of intact cells Requires dissociated tissue for single cell analysis, limited to static timepoints, and limited access to facilities with ideal capabilities [ 16 , 19 , 27 , 65 ] …”