Mitochondria are the proverbial 'powerhouse' of eukaryotic cells producing a bulk of their energy supply in the form of adenosine triphosphate (ATP). As dynamic organelles, mitochondria can modify their architecture - sometimes taking up as much as 25% of a cell's volume - to accommodate for the cell's metabolic needs and other cellular processes, including signaling, cellular differentiation, cell cycle maintenance, cell growth, and apoptosis. In immunity, the importance of mitochondrial dynamics is apparent, as many pathogens have evolved mechanisms to modulate host cell mitochondrial remodeling and function to promote their survival. The retrograde signaling initiated by dysfunctional mitochondria can give rise to global changes in gene expression that affects cell morphology and function and initiates the propagation of disease processes. Alterations in mitochondrial morphology and function are good indicators of cell health, and multiplexing mitochondrial morphology reagents with probes that assess function can provide more in-depth information about mitochondrial health. AAT Bioquest has developed a wide range of mitochondrial stains and functional reagents to investigate mitochondria in live- and fixed-cell imaging applications. In this article, we highlight a few of our most-cited probes.

Mitochondria morphology is complex, dynamic, and highly varied. Through tightly regulated fission, fusion, and mitophagy events, mitochondria can appear in various forms within a cell. From fragmented morphologies of small spheres and short to elongated tubules to a reticulated morphology in which the mitochondrion is a single network of many-branched structures. The number of mitochondria present and their location in a cell can also vary and depends on several variables, including cell or tissue type, developmental stage, metabolic requirement, and overall cell health. For instance, liver hepatocytes, which are involved in metabolism, detoxification, and protein synthesis, have about 1000-2000 mitochondria per cell. Because alterations in mitochondrial function result in dramatic modifications to its structure, staining mitochondria using fluorescent dyes such as MitoLite™ dyes and visualizing their morphology through a microscope can provide significant information about their overall biology, localization, and functional state (Table 1). Figure 1 shows HeLa cells with both spheroid-shaped mitochondria and mitochondria with normal reticulated morphology.

Mitochondrial dysfunction - characterized by an inadequate number of mitochondria, an inability to provide necessary substrates to mitochondria, or the loss in efficiency of their electron transport chain and reductions in ATP production - is a hallmark of cellular toxicity and is associated with many chronic diseases. These include neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, cardiovascular diseases, diabetes, metabolic disorders, autoimmune diseases, gastrointestinal disorders, fatiguing illnesses, musculoskeletal diseases, and chronic infections. AAT Bioquest provides a variety of functional tools for studying mitochondrial function from various perspectives, including probes for mitochondrial membrane potential, autophagy/mitophagy, oxidative phosphorylation, calcium flux, and cytosolic pH (Table 2).

Multiplexing mitochondrial functional tools with morphology probes can significantly improve the study of mitochondria. For example, MitoLite™ Green FM can be combined with the mitophagy-sensitive dye Mitophagy Red™ to monitor the mitochondria-lysosome interactions vital for maintaining homeostasis in eukaryotic cells. These include mitochondria-lysosome fusion, a process that selectively removes redundant or damaged mitochondria (Figure 3), or mitochondria-lysosome contact (MLC). MitoLite™ dyes can be combined with potential-dependent probes such as TMRE or TMRM to monitor mitochondrial structural integrity while also assessing mitochondrial membrane potential, as well as with the mitochondrial calcium indicator Rhod-2 am or with mitochondrial ROS sensors.