Proceedings of The Physiological Society

Mitochondria: Form and function (London, UK) (2017) Proc Physiol Soc 38, PC03

Poster Communications

Serial Block Face Electron Microscopy Reveals Distinct Features of Mitochondrial Form in Striated Muscle

E. Cocks1, K. White1, M. Taggart1

1. Newcastle University, Newcastle Upon Tyne, United Kingdom.

Introduction Mitochondria are important organelles for fuelling cellular function. They are known to have malleable morphologies that may relate to their involvement in pleiotropic cell functions and possibly mitochondrial-related diseases. However, analysis of the structure of mitochondria, and their positioning as a ‘network', would benefit from extending examinations beyond traditional spatial resolutions of fluorescent confocal microscopy or single section transmission electron microscopy (TEM). With the development of Serial Block Face Scanning EM (SBFSEM), it is now possible to analyse and reconstruct mitochondria and surrounding elements in the x, y and z dimensions and at nm resolution. The purpose of this study was to investigate the range of whole mitochondrion morphologies evident in skeletal muscle cells. Method The psoas and soleus muscle from adult guinea pigs (killed according to Home Office guidelines) were collected. The samples were processed using a SBFSEM heavy metal protocol (Deerinck, 2010) and prepared for examination within the SBFSEM. The samples were imaged at a low magnification (2-4kx) with varying image size (2000-3000pixels) and resolution (8-19nm), and at least 250 serial sections (70nm) taken. The raw data was analysed manually in Microscopy Image Browser (MIB) and subsequent segmentations were reconstructed in 3D using Amira. This type of analysis is time consuming so only a portion of the visualised mitochondria was segmented. These were selected randomly by placing a grid (5 to 10µm2) over the series of images. Then each whole mitochondrion that crossed the grid, on a slice chosen randomly, was reconstructed. Results Visualisation of the raw data showed that mitochondria, both sub-sarcolemmal and inter-sarcomeric, could extend over 50 to 150 images, indicative of mitochondria having a range of volumes from 0.05 to 5.03μm3 (psoas) and 0.06 to 8.06 µm3(soleus). Several general morphologies were categorised as follows: columnar, spheroidal, framework or irregular (Figure 1). In addition, mitochondrial branching features were noted that varied in complexity from no branches, singular or multiple segments to those of horseshoe or doughnut appearance. In psoas muscle cells there appeared to be more columnar mitochondria (71+12%) than in the soleus (17+14%, p=0.06, n=3, mean+sem, student's unpaired t-test), although it did not reach significance. Conclusion In summary, SBFSEM is a powerful technique for examining individual mitochondrion structure. We have found that there are distinguishable morphologies of mitochondria in skeletal muscle and a classification system has been developed to analyse these. These approaches will inform further studies investigating mitochondrial morphological changes with development and disease.

Where applicable, experiments conform with Society ethical requirements