GlowMito - Red fluorescent mitochondrial tracker

GlowMito was derived from a triamino-phenazinium belonging to the induline family. With their attractive absorption properties and excellent stability, these molecules have a long history as dyestuffs and attracted major attention in applications from photoreceptor or electrophotography agents to ingredient in color industry.

These derivatives also showed great potential to serve as new mitochondrial probes, although biological applications had so far been neglected. In particular, their appealing photophysical properties characterized by high fluorescence quantum yield and absorption cross-section make them ideal candidates for two-photon excitation microscopy. After having modified some of their substituents to make them internalized by cells and mitochondria more efficiently, GlowMito was born!

The newly synthesized compound, soluble in water, proved very efficient at quickly accumulating within mitochondria without inducing any cytotoxic effects or altering normal mitochondrial functions. Since then, GlowMito expanded the scope of mitochondrial probes by providing a solution of choice to label the entire mitochondrial network quickly and safely in live cells, deep tissues, and even anaerobic organisms.

GlowMito is a cell-permeant probe that is rapidly internalized by cells and specifically target the mitochondria. The quick GlowMito penetration in cells was made possible by the introduction of lipophilic substituents in the original molecule, coupled with its cationic charge, thereby enhancing passive diffusion across cell membranes as it is observed for various lipophilic cations.

As known for various other lipophilic cations, GlowMito accumuation within mitochondria is at least partly driven by membrane potential. Yet, although the precise mechanisms are not yet unraveled, it is likely that GlowMito also relies on other membrane potential-independent mechanisms such as specific interactions with anionic species within the mitochondrial matrix, enhancing the retention of the dye within the mitochondria even with reduced potential. 

The strong ability of GlowMito to specifically target mitochondria has been meticulously checked by co-localization studies. More generally, the ability of lipophilic cations to specifically target mitochondria is already well-established and conjugating molecules to lipophilic cations is a commonly used method to develop mitochondria-targeted compounds.

Contrary to classical potential-dependent probes, GlowMito is minimally affected by membrane potential loss and will still produce a visible signal in mitochondria with poor membrane potential. This was demonstrated by pilot studies, later confirmed by early users, showing that GlowMito signal was higher than that of TMRM in presence of an uncoupler. One of our early users even found that GlowMito was able to label hydrogenosomes in the anaerobic organism Trichomonas vaginalis, that have only little -if any- membrane potential, and for which other probes like JC-1, DioC6 and TMRE were not working. 

GlowMito produces a bright, red labeling of the entire mitochondrial network, including those with reduced potential. It is perfectly suitable for:

·       Live imaging: study of mitochondrial dynamics (velocity, localization), structural changes, multiplexing (potentiometric dyes, calcium signaling probes, etc)

·       Downstream analysis: flow cytometry, oxygraphy, etc

We do not recommend its use to measure mitochondrial mass or volume density.

No, GlowMito can be used to label mitochondria in live samples only and is not retained after aldehyde fixation.

So far, GlowMito has been successfully used to label mitochondria in the following biological samples:

·       Human cells: HEK293, HeLa, MCF-7, MDA-MB-231, HMLE, UACC-62, U2-OS, Gli36, HAEC, SH-SY5Y, A-172, A549, patient-derived skeletal muscle cells, primary smooth muscle cells & iPSC-derived cardiomyocytes

·       Monkey cells: COS-7

·       Mice cells: primary cortical neurons

·       Tissues: hiPSC-derived heart tissues & mice isolated pressurized blood vessels

·       Parasitic protists: Trichomonas Vaginalis

GlowMito showed no internalization in yeast cells

We would greatly appreciate your valuable inputs by helping us complementing this list with any of your positive research experiences. Drop us a message through our Contact page!

GlowMito comes as a 1 mM stock solution in water, that you can directly dilute in your culture medium. All you need to have on your side is a culture vessel, pre-heated culture medium or your choice and your biological sample.

The full excitation (solid line) and emission (dotted line) spectra of GlowMito in one-photon microscopy is presented below (Ex:542nm/Em:690nm): 

For use in flow cytometry, we recommend using a 488nm laser and a 655-730 nm emission filter. Its wide emission spectrum makes GlowMito compatible with a wide range of standard and complex cytometers (including BD Accuri, FACSCanto, LSRFortessa, FACSAria, FACSMelody, FACSymphonie, Cytek Aurora). 

The absorption spectrum of GlowMito in two-photon microscopy is presented below (red line, cross-section = 224GM at 790nm):

The total procedure to label mitochondria in live cells should take no more than 40 min. For deep tissues, total labeling time might need to be extended up to 1.5 h. 

The GlowMito signal has been shown to remain stable & specific for up to 3 days with no sign of cell toxicity.  

The 1 mM GlowMito stock solution can be stored at 4°C for one month. For long-term storage, aliquot to avoid repeated freeze/thaw cycles and store at -20°C for up to 6 months. Always store GlowMito protected from light.

One vial contains 125µL of a 2,000X aqueous solution for a final volume of 250mL of imaging medium when used at the recommended working concentration. The optimal GlowMito working concentration might need to be increased up to 2-fold when working with deep tissues.

GlowMito can be prone to photobleaching when exposed to high-intensity and/or prolonged illumination in one-photon confocal microscopy. Keep in mind that GlowMito photobleaching should be minimal when imaged in 2-photon microscopy.

Here are a few tips you can implement to reduce GlowMito photobleaching:

·       Adjust laser intensity and keep it to a minimum.

·       Reduce exposure time and try to increase intervals between acquisitions where possible.

Note: capturing a series of short exposure images at regular intervals, instead of a single long exposure, can be used to assess mitochondrial dynamics over time.

·       If not sufficient, using anti-fading agents will reduce photobleaching during prolonged imaging sessions.

Note: the ProLong Live antifade reagent has been successfully used in combination with GlowMito and enabled strong conservation of GlowMito signal after 15 sec of continuous light exposure on a confocal microscope.