Imaging and Analysis of Mitochondrial Dynamics in Living Cells
One of the most striking features of plant mitochondria when visualized in living tissue is their dynamism. The beauty of cytoplasmic streaming, driving, and being driven by the motility of mitochondria and other small organelles belies the complexity of the process. Equally, capturing that dynamism and investigating the genes, proteins, and mechanisms underpinning the processes using molecular cell biology and bioimaging is a complex process. It requires the generation of fluorescent protein constructs, stable transgenic plants sometimes expressing multiple fusions, and generation of mutants, even before one is ready for analytical experimentation. Here, we describe some of the key tools and methods necessary to investigate plant mitochondrial dynamics.
Key wordsMitochondria Image analysis Microscopy Deconvolution Tracking Motility Colocalization Live cell imaging
The Olympus microscope system in the Logan lab at the University of Saskatchewan, Canada, was purchased with a Canada Foundation for Innovation LOF grant to DCL. Imaris and AutoQuant were purchased with an NSERC RTI award to DCL. The Zeiss microscope system in Angers, France, was purchased by SFR QUASAV as part of the Campus Végétal project. Thanks to David Macherel for supporting my move to Angers and his continued support thereafter, and for alerting me to the ultrathin tape. AME was supported by an NSERC Discovery grant to DCL, by the Department of Biology at the UofS, and UGF and GTF awards. DSBE was supported by NSERC Discovery grants to DCL and Dr. Chris Todd (Biology, UofS).
Movie showing the mitochondria as imaged (in green) and identified (as red spots) by Imaris. The movie was captured at 1 fps and runs at 2 fps and thus the whole movie represents 30 s in real time (MOV 909 kb)
As movie 1 but displaying the last stage of the spot tracking process where each spot (mitochondrion) is connected to its movement track. Any spots (mitochondria) that are not tracked have been eliminated from processing by the chosen algorithm (Subheading 3.7) (MOV 938 kb)
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