Mitochondria are essential for maintaining cellular health, with a key aspect of mitochondrial function being the mitochondrial membrane potential (mitochondrial potential). Mitochondrial potential is a valuable biomarker for detecting any disruptions linked to mitochondrial dysfunction. This is because it reflects the electrical gradient central to processes like ATP synthesis and calcium homeostasis across the inner mitochondrial membrane. These aspects make mitochondrial potential a useful metric for following apoptosis.
Rhodamine-based fluorescent probes such as TMRM, TMRE, and JC-1 are commonly used to measure mitochondrial membrane potential and work by accumulating within mitochondria in a mitochondrial potential-dependent manner. However, they have limited spectral compatibility and can reflect interference from factors other than mitochondrial potential (e.g., oxidative stress with JC-1). MitoView™ 633, part of Biotium’s MitoView™ catalog, is a far-red fluorescent dye specifically designed for mitochondrial imaging that has now been shown to have great potential in multiplexing and ΔΨm measurement, offering a promising imaging alternative to TMRM.
In a 2023 publication in Frontiers Physiology, Ernst et al. systematically characterized MitoView™ 633 and its performance in comparison to TMRM. Its spectral properties, mitochondrial staining specificity and photostability were evaluated, as well as its performance during mitochondrial potential depolarization and compatibility with other fluorescent markers. MitoView™ 633’s far-red emission reduces light scattering and enhances tissue penetration, making it advantageous for imaging in deeper tissue layers, and spectral analysis showed that it had superior thermal stability compared to TMRM. Confocal microscopy confirmed that MitoView™ 633 localized specifically within the mitochondrial matrix and exhibited minimal photobleaching, making it well-suited for extended imaging sessions. The dye also showed compatibility with co-staining alongside MitoSOX™ Red and Fluo-4 AM, enabling simultaneous imaging of complex mitochondrial and cellular processes in intact cells.
MitoView™ 633 was found to be a robust, versatile, and reliable tool for live-cell imaging of mitochondrial membrane potential, with unique spectral properties that enhance its compatibility with other dyes and its suitability for imaging in deeper tissues. Additionally, MitoView™ 633 proved advantageous for simplifying experimental workflows and reducing preparation time without requiring a washing step after dye loading. These features position MitoView™ 633 as a robust, versatile tool for studying mitochondrial dynamics and mitochondrial potential in live cells, with potential applications in both physiological and pathological contexts.
Learn more about Biotium’s wide range of mitochondrial dyes, in a wide variety of colors. Biotium also offers MitoView™ Fix 640, a far-red mitochondrial stain suitable for no-wash, long term staining in live cells that is well-retained after fixation, permeabilization, and subsequent immunofluorescence staining. Biotium’s Mitochondrial Marker Antibodies stain mitochondria in fixed cells or tissue sections and are available with a wide selection of bright and photostable CF® Dyes and other conjugations.
Full Citation
Ernst P, Kim S, Yang Z, Liu XM and Zhou L (2023) Characterization of the far-red fluorescent probe MitoView™ 633 for dynamic mitochondrial membrane potential measurement. Front. Physiol. 14:1257739. doi: 10.3389/fphys.2023.1257739