Furthermore, we discussed the potential of automatic mitochondrial segmentation, classification and prediction of mitochondrial abnormalities using machine learning techniques. We listed and applied pipelines and packages available in ImageJ/Fiji, CellProfiler, MATLAB, Java, and Python for the analysis of fluorescently labeled mitochondria in microscopy images and compared their performance, usability and applications. We reviewed the current image-based analytical tools and machine-learning techniques for phenotyping mitochondrial morphology in different cancer cell lines from confocal microscopy images. The mitochondrial dynamics is related to the initiation, migration, and invasion of diverse human cancers and thus affects cancer metastasis, metabolism, drug resistance, and cancer stem cell survival. ![]() Depending on the environmental conditions, the mitochondrial morphology dynamically changes to match the energy demands. Mitochondria are dynamic organelles that integrate bioenergetics, biosynthesis, and signaling in cells and regulate redox homeostasis, apoptotic pathways, and cell proliferation and differentiation. Mitochondria are shown in a fragmented state during cytosolic release of pro-apoptotic signaling factors and related to a swollen stated upon loss of ΔΨm (gradient arrow). For example, DR activation activates pro-apoptotic tBid, which leads to Bax activation at the mitochondria. The scheme summarizes the subcellular impact of our drug selection and depicts the three possible morphologic states of mitochondria: networked, fragmented and swollen. MCF-7 stably expressing Mito-GFP were incubated for 6 hours at 37☌ with 7 different apoptotic drugs inducing a variety of cellular stress: calcium overload (thapsigargin, 1 µM) DNA synthesis inhibition (camptothecin, 2 µM) ATP synthesis inhibition (oligomycin, 10 µM) death receptor (DR) pathway activation (TNFα, 43 ng/mL and TRAIL, 20 ng/mL) mitochondrial fragmentation (ceramide, 300 µM) as well as a mitochondrial uncoupler (CCCP, 20 µM). B) Cartoon scheme representing the tested apoptotic drugs and its targets. These values (N/F/S) can be averaged per cell to obtain whole cell population shifts on mitochondrial morphology under the tested condition. Percentage values of Networked/Fragmented/Swollen (N/F/S) mitochondria are attributed to each cell. Here we present examples for 6 hours incubation (37☌) in control condition (FM) and drug conditions (ceramide, 300 µM, camptothecin, 2 µM). Population wide analysis of mitochondrial morphology.Ī) CellProfiler pipeline applied to “Full Images”- (i) Representative examples of images as obtained by the DVRT microscope after deconvolution (ii) Cell border identification and segmentation with final classification for each considered cell (color code corresponds to single cell segmentation) (iii) Mitochondrial segmentation per cell (color code corresponds to single mitochondrion segmentation).
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