Quantification of Mitochondrial Morphology in situ

The structural organization of mitochondria reflects their functional status and largely is an index of cell viability. The indirect parameter to assess the functional state of mitochondria in cells is the degree of their fragmentation, i.e., the ratio of long or branched mitochondrial structures to round mitochondria. Such evaluations requires an approach that allows to create an integral pattern of the three-dimensional organization of mitochondrial reticulum using confocal images of mitochondria stained with a fluorescent probe. In the present study, we tested three approaches to analyzing the structural architecture of mitochondria under normal conditions and fission induced by oxidative stress. We revealed that, while the most informative is a three-dimensional reconstruction based on series of confocal images taken along the Z-dimension, with some restrictions it is plausible to use more simple algorithms of analysis, including one that uses unitary twodimensional images. Further improvement of these methods of image analysis will allow more comprehensive study of mitochondrial architecture under normal conditions and different pathological states. It may also provide quantification of a number of mitochondrial parameters determining the morphofunctional state of mitochondria—primarily, their absolute and relative volumes—and give additional information on threedimensional organization of the mitochondrion.