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Scalable Fluorescence Microscopic Assays of Organelle Transport and Mitochondrial Swelling
Author(s) -
Gerencser Akos A,
Nicholls David G,
Brand Martin D
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.775.2
Subject(s) - organelle , swelling , biophysics , fluorescence , mitochondrion , cytoplasm , fluorescence lifetime imaging microscopy , biological system , materials science , chemistry , biology , microbiology and biotechnology , physics , optics , composite material
Fluorescence microscopic tools are widely used for assessing mitochondrial biology, however quantitative assays of parameters such as organelle dynamics are not trivial. High‐content imaging systems established the need for scalable assays that are robust and are able to evaluate biological parameters in an automated, unsupervised manner. We introduce here a software platform implementing novel image processing techniques allowing low light level but robust determination of mitochondrial swelling and organelle motion in cultured cells. Mitochondrial swelling is measured by the ratio of fluorescence intensities in high over low frequency spatial band pass filtered copies of the same image. This ratio is highly sensitive to mitochondrial swelling, but insensitive to fission or fusion, motion and overlaps of mitochondria. Organelle transport is assayed by optical flow, featuring instantaneous velocity determination from a pair of images by detecting motion of edges. Optical flow provides velocity vectors; therefore anterograde, retrograde transport and local, wiggling motion can be distinguished. Both assays are scalable, applicable for imaging in microplates and are accessible through a user friendly interface.