3.1.

Optimizing Live Plasma Membrane Imaging with MemBright Probes

In order to be able to place the molecules within the cell shape, and in collaboration with chemists, we have selected new membrane probes capable of revealing the cell shape and imaging fine structures, such as dendritic spines.²³

These membrane probes, named MemBright, upon insertion in the membrane, emit fluorescence with narrow emission peaks, allowing correlation with other conventional fluorescence for multi-color labeling. A family of seven members is now available and can be used all over the fluorescence spectrum (from 480 and 750 nm). Upon incubation with living cells, these probes insert directly into the membrane through a lipid anchor and thus reveal the cell shapes without the use of any transfection or viral vectors (Fig. 2). This strategy thus makes it possible to reveal all neurons and/or glial cells in a few minutes without any toxicity.

Fig. 2

Confocal image (20×) of hippocampal neurons after 2 months in a culture labeled with Cy3.5-MemBright probe reveals dendritic and axonal branch complexity. Scale bar: $100\mu \mathrm{m}$.

The big advantage of MemBright probes is also its fluorogenic property. Indeed, MemBright probes are non-fluorescent within media and become fluorescent when reaching the plasma membrane. It means that the probe can be let within the cell culture media without having a fluorescence background under the microscope. Letting the probe in the cell chamber during live acquisition allows the perpetual replacement of the probes if bleaching occurs, thus leading to persistent bright labeling of the membrane.

MemBright probes are more efficient when incubated on cells in the absence of serum to avoid any titration of the probes by serum fat. In neurons and glial cells, we usually incubate MemBright probes in Krebs Ringer solution at a concentration of 200 nM at 37 deg under the microscope. The absence of serum optimizes the labeling, and the absence of phenol red lowers the fluorescence background. Fluorescence on the plasma membrane appears very fast within a few minutes. To avoid saturation of the plasma membrane with a huge amount of lipids, it is crucial not to use a high concentration of probes. MemBright probes are sufficiently bright to be used at the nanomolar range, whereas other commercial probes have to be used at the micromolar range. Moreover, it should be stressed that illumination of any fluorescent probes may induce the production of reactive oxygen species that can imbalance the intracellular redox state and be deleterious to the cell.²⁴ Thus, it is also a good practice to minimize illumination time and frequency, to the minimal amount needed to the right sampling of a biological event. Previously, we could follow neuronal growth over time during 13 h, imaging every 2 min with low laser power (0.2% of an 561 laser line of an Elira PS1), without any detrimental effects.²³ At last, we have shown that MemBright probes are resistant to permeabilization when fixed properly with a mixture of 4% paraformaldehyde-0.2%glutaraldehyde, and can thus be combined with conventional immunolabeling with primary and secondary antibodies.²³ MemBright staining can be correlated either to live antibody staining or with immunochemistry on fixed samples. We could show double labeling of the plasma membrane and live L1-CAM endocytosed antibodies. We also showed that intracellular vesicular transporter VGLUT could be revealed by immunochemistry within 3D neuronal cell shape reconstructed using MemBright. This property thus allows identifying surface or internal protein locations using MemBright counterstaining to visualize cell shape. We have selected MemBright probes for their stability at the plasma membrane and their slow endocytosis. However, on long-term incubation, they will be finally endocytosed and can thus be used to label endocytic pathways [Fig. 3(c)].²⁵

Fig. 3

Confocal images (93×) of various cell types labeled with Cy3.5-MemBright probe. (a) 3D rendering of E.coli bacteria incubated overnight with MemBright probes. (b) 3D rendering of HeLa cells incubated 30 min with MemBright. (c) Confocal section of hippocampal neurons incubated several hours with MemBright, revealing intracellular vesicles.

3.2.

Imaging Plasma Membrane of Various Cell Types

We originally showed that MemBright probes could be used in various cell types, such as epithelial cells in culture (HeLa cells or KB cells),^23,26–28 and dissociated hippocampal neurons,²³ hippocampal astrocytes.²³ We could also use MemBright probes to label live brain (hippocampus, cortex, and cerebellum) or liver slices, allowing the labeling in depth and imaging using confocal or two photons microscopy.²³ Since our first paper in 2019, MemBright probes have been used by several other labs and cited in more than 60 articles and 39 reviews.^29–57 It has been used in B lymphocytes,⁵⁸ in A431 cells,⁵⁹ and reused in neuronal cells to label growth cone and initial segments of hippocampal neurons,⁶⁰ presynaptic terminals,²⁹ and post-synaptic compartments.⁵³ It has also been used to label apoptotic bodies (AB), microvesicles (MV), and small EV (sEV) isolated from MIN6 pancreatic beta cells exposed to inflammatory, hypoxic, or genotoxic stressors.⁶¹ Since we were asked several times if MemBright could be used on bacteria, we did recently E. coli live labeling with MemBright – CY3.5. As shown in Fig. 3(a), the bacteria 3D shape can be efficiently labeled in live.

3.3.

Imaging Extracellular Vesicles

MemBright has been widely adopted by the extracellular vesicles community⁵¹ to track extracellular vesicles both in vitro or in vivo^61–81 in hippocampal⁶³ or cortical neurons,⁷⁹ zebrafish,^62,67,72 breast cancer cells or tumours,^66,71,80 myotubes,⁸² and red blood cells.^74,76

Particle size distribution and zeta potential analysis of EVs derived from A375 cells using nanoparticle tracking analysis (NTA) showed that EVs labeled before and after labeling by MemBright have almost no change in size and only a slight shift of zeta potential.⁷⁵ Due to its ease of use and brightness, MemBright has thus been widely used to label exosomes. However, it should be stressed that MemBright is not specific to extracellular vesicle labeling. MemBright will be able to label any membrane in contact with the probe. That means that membrane debris trails left behind by migrating cells will be labeled, whatever the nature of the membrane (EVs or not). Any membranous organelles (tubules, endosomes, lysosomes, synaptosomes, etc.) that would have been retrieved by ultracentrifugation can be labeled when incubated with MemBright. Some controls are thus needed before labeling to ensure that the fraction is homogeneous and not contaminated by different organelles.

Hyenne et al.⁶² show that MemBright can be used in pulse-chase experiments and that some CD63-GFP EVs can be labeled with MemBright, while others are not.⁶² Sung et al. concluded that MemBright can label exosomes as well as plasma membrane-derived EVs, but that MemBright does not label all exosomes.⁴⁷ Using a pulse-chase experiment, it is expected that not all endosomal compartments will be labeled. Indeed, only those deriving from the plasma membrane exposed to the MemBright at a time $t$ will be visible. It is likely that a proportion of EVs that were generated before or after incubation with MemBright and that are then stored in the cell will not be labeled by the MemBright wave. It is therefore essential to properly calibrate the labeling time incubation and the chase time to observe the desired events.