PKH dyes, which are currently the most widely used fluorescent probes for extracellular vesicle (EV) labeling, have some limitations. For example, these dyes tend to aggregate, leading to formation of EV-like nanoparticles that can be taken up by cells. Moreover, it has been suggested that PKH dyes trigger an enlargement of EVs because of membrane fusion or intercalation. To overcome these limitations, we developed three novel extracellular vesicular-membrane-binding fluorescent probesMem dye-Green, Mem dye-Red, and Mem dye-Deep Redfor monitoring EV uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold and amphiphilic moieties on the cyanine. The three dyes have different photophysical characteristics. To investigate the characteristics of the Mem dyes for EV labeling, we performed nanoparticle tracking, zeta potential measurements, and confocal microscopy. The dyes enable highly sensitive fluorescence imaging of EVs. They can also be used to observe EV dynamics in live cells. The Mem dyes show excellent EV labeling with no aggregation and less particle enlargement.

As a process of cellular uptake, endocytosis, with gradient acidity in different endocytic vesicles, is vital for the homeostasis of intracellular nutrients and other functions. To study the dynamics of endocytic pathway, a membrane-anchored pH probe, ECGreen, is synthesized to visualize endocytic vesicles under structured illumination microscopy (SIM), a super-resolution technology. Being sensitive to acidity with increasing fluorescence at low pH, ECGreen can differentiate early and late endosomes as well as endolysosomes. Meanwhile, membrane anchoring not only improves the durability of ECGreen, but also provides an excellent anti-photobleaching property for long-time imaging with SIM. Moreover, by taking these advantages of ECGreen, a multidimensional analysis model containing spatial, temporal, and pH information is successfully developed for elucidating the dynamics of endocytic vesicles and their interactions with mitochondria during autophagy, and reveals a fast conversion of endosomes near the plasma membrane.

We have developed three types of exosomal membrane binding fluorescent probes, Mem Dye-Green, Mem Dye-Red and Mem Dye-Deep Red, to monitor exosome uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold, which allows for highly sensitive fluorescence imaging of the exosome. These dyes can also be used to observe the dynamics of exosomes in live cells. The use of PKH dyes (Figure 1), which are currently the most widely-used fluorescent probes for exosome labeling, has some limitations. For example, PKH dyes tend to aggregate to form exosome-like nanoparticles, and these nanoparticles are uptaken by cells. Moreover, Mehdi suggested that the use of PKH dyes triggers an enlargement of the exosome size owing to membrane fusion or intercalation. To overcome the limitations of PKH dyes, we introduce amphiphilic moieties to the cyanine. To investigate the characteristics of the Mem Dyes as exosome labeling probes, we perform nanoparticle tracking analysis (NTA), zeta potential measurement and confocal microscopy. The Mem Dyes show excellent performance for exosome labeling (no aggregation and less size shift).

The plasma membrane (PM) plays a critical role in many cellular processes, and PM dysfunction is a key biomarker related to the cell status and several diseases. Imaging techniques using small fluorescent probes have become increasingly important tools for visualizing living cells, particularly their PMs. Among the commercially available PM-specific probes, PKH dyes are widely used; however, the utility of these dyes is limited by their short membrane retention times and high cytotoxicity. Herein, PlasMem Bright Green and Red are implemented as new PM-specific fluorescent probes, which employ a polycyclic aromatic fluorophore to improve their retention ability and a strong acid moiety to reduce their transmembrane diffusion and cytotoxicity. We demonstrate that the long retention and low cytotoxicity of the PlasMem Bright dyes enable them to be applied for observing neuronal PMs and monitoring PM dynamics involving the endocytic pathway. Furthermore, we successfully detected mitochondria in nerve axons over long periods using PlasMem Bright dyes. Finally, the combined use of exosome staining probes and PlasMem Bright dyes allowed clear visualization of the endocytic pathway.TOC graphic

Endocytosis involves plasma membrane-derived vesicles for the recycling of intra- and extracellular components. Increasing evidence suggests that endocytosis is related to maintaining intracellular homeostasis and defense against disease. Consequently, investigation of the endocytic pathway attracts considerable scientific interest. This study reports live-cell imaging of endocytosis using the newly-developed fluorescent probe ECGreen. We demonstrate that ECGreen is not membrane permeable and its fluorescence signal increases in acidic conditions. Because of these characteristics, ECGreen remains on the plasma membrane, and then shows increased fluorescence when it is internalized into the acidic vesicles formed in the endocytic process. ECGreen allows direct observation of the internalized vesicle; it is a valuable new probe for endocytic imaging.