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Membranes Probes

Mise à jour 17 janvier 2018

Fig. 2. Examples of environment-sensitive membrane probes developed in our group. Probe PA was developed together with Yosuke Niko (Japan).

We develop new molecular probes sensitive to different properties of lipid membranes. The selectivity of the probes to a particular membrane property can be achieved by proper selection of the fluorophore and its appropriate localization in the lipid membrane (3). Representative examples are probes of hydration and surface charge, F2N12S 1, transmembrane potential, di-SFA 2 and lipid order : F2N12S 1, NR12S 3, bNR10S 4 and PA 5.

Apoptosis detection

Fig. 3. Response of a polarity-sensitive membrane probe F2N12S to apoptotic changes in cell plasma membranes. Changes in the ratio of two emission bands of F2N12S on apoptosis in CEM cells imaged by confocal microscopy. Data adapted from 1.

Normal cell membranes exhibit strong transbilayer asymmetry, while in apoptotic cells, it is lost. In our earlier work in 2007, within a group of Yves Mely (UMR7213, Strasbourg), we developed F2N12S, which incorporates selectively into the outer leaflet of the cell plasma membrane, can detect this loss of asymmetry by changing its N*/T* intensity ratio 1. Being ratiometric, the response of the new probe can be easily quantified by microscopy techniques, a feature that can be hardly achieved with the commonly used fluorescently labeled annexin V. Later on, we showed that NR12S can also be applied for apoptosis detection 6.

Probes for lipid order and microdomains

Fig. 4. Ratiometric imaging of giant vesicles composed of Lo and Ld domains as visualized by NR12S (left). Intact cells and cells after cholesterol extraction with MβCD showing different color in the ratiometric images (middle and right). Two-photon excitation at 830 nm was used. Sizes of the images were 50x50 μm. Data from Ref 3.

Membrane domains or “rafts” are believed to be involved in various cell functions. In model membranes, these domains, defined as liquid-ordered (Lo) phase enriched in cholesterol (Chol) and sphingomyelin (SM), can be observed as “rafts” floating in the liquid disordered (Ld) phase, enriched in unsaturated phospholipids (DOPC). According to our earlier work in 2010, probe NR12S due to its high sensitivity to the membrane hydration allows visualization of lipid domains in model membranes and monitoring lipid order in cellular membranes using ratiometric imaging (Fig. 4) 3. Later on, we developed a bulky version of NR12S, so-called bNR10S, and BHQ-2 based quencher, which showed specific partitioning to Ld phase. These probes enabled direct demonstration of phase heterogeneity in plasma membrane of living cells 4.

Fig. 5. Ratiometric (a-b) and FLIM (c-e) imaging of lipid order in living HeLa cells using solvatochromic probe PA. The pseudo-colors represent the ratio of the long- to short-wavelength emission channels (550-700 nm to 470-550 nm). (b) Calibration of the ratio using suspension of lipid vesicles representing Ld (DOPC) and Lo (SM/Cho, Lo phase) phases. (c) FLIM image of HeLa cells and (d) zoom on the region of interest. (e) Calibration images of suspensions of vesicles. Adapted from 6.

In more recent studies, together with Yosuke Niko, JSPS post-doctoral fellow in our group, we showed that push-pull pyrene (PA, Fig. 2), similarly to Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties 5. In addition to red-shifted absorption compatible with 405-nm diode laser, PA shows higher brightness and much higher photostability than Laurdan. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting Lo and Ld phases. PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging and FLIM using PA revealed variation of lipid order within different cellular compartments : plasma membranes are close to Lo phase, while intracellular membranes are much less ordered, reflecting lower cholesterol content (Fig. 5) 5. PA probe constitutes thus a new powerful tool for biomembrane research.

Bright membrane probes

Our ongoing work on development of membrane probes revealed the other important aspect – the need in the bright probes that can specifically stain the cell plasma membranes in a desired color. In this case, one needs to use fluorophores with high brightness and photostability, exhibiting narrow absorption and emission bands in the regions complementary to commonly used fluorescent proteins. Here, we considered two blue and far red spectral regions. Multi-color imaging needs these probes, because of their complementary with green and red fluorescent proteins. Using 3-methoxychromone dyes, we designed two blue membrane probes F2N12SM and FC12SM (Fig. 6) exhibiting >100-fold fluorescence turn-on (fluorogenic response) on membrane binding, large Stokes shift (70–90 nm) as well as high brightness and photostability 7. These unique properties enabled cellular imaging at low probe concentrations (20–50 nM) with minimal background from cell auto-fluorescence and from free probe. RGB multicolour imaging was successfully realized using these probes in combination with common green and red markers (Fig. 6). These new blue probes may fill the gap in multi-color microscopy.

Fig. 6. (Left) Chemical structures of the new membrane probes, F2N12SM and FC12SM. (Right) Fluorescence confocal images of HeLa cells without probes (A) and stained with F2N12S (B), F2N12SM (C) or FC12SM (D) at 50 nM concentrations. (E-H) Multicolour confocal imaging using F2N12SM (blue, E), LysoTracker® Green DND-26 (green, F) and mCherry (red, G) and merge of the three images (H). Scale bar is 20 µm. Adapted from 7.

To obtain bright far-red membrane probes we focused on squaraines, dyes exhibiting exceptional extinction coefficient ( 300,000 M-1 cm-1) as well as high quantum yield and photostability. We designed three amphiphilic squaraine probes containing hydrocarbon chains and zwitterionic polar head groups 8. We found that SQ8S quickly crossed the plasma membrane to efficiently stain the endoplasmic reticulum membranes (Fig. 4). SQ12S distributeed in both plasma and ER membranes (Fig. 4). Finally, dSQ12S, due to its two anchors, stained specifically plasma membranes without significant internalization (Fig. 4). Remarkably, we were able to obtain high quality confocal images even at 1 nM of dSQ12S and SQ8S, whereas commercial dye DiD failed to stain plasma membrane even at 20 nM concentration (Fig. 4) 8. To the best of our knowledge, SQ8S and dSQ12S, are respectively the brightest molecular ER and plasma membrane fluorescent probes developed to date.

Fig. 7. (Left) Chemical structures of squaraine probes. (Right) Laser scanning confocal microscopy images of HeLa cells incubated for 5-10 minutes with the DID or squaraine probes at 20 nM without washing (red). Nucleus was stained by Hoechst (2 µg.mL-1) (blue) and the plasma membrane was stained with WGA-488 (5 µg.mL-1) (green). Scale bar is 10 µm. Adapted from 8.


1) Shynkar, V. V. ; Klymchenko, A. S. ; Kunzelmann, C. ; Duportail, G. ; Muller, C. D. ; Demchenko, A. P. ; Freyssinet, J. M. ; Mely, Y. : Fluorescent biomembrane probe for ratiometric detection of apoptosis. Journal of the American Chemical Society 2007, 129, 2187-2193.

2) Klymchenko, A. S., Stoeckel, H., Takeda, K., and Mely, Y. (2006) Fluorescent probe based on intramolecular proton transfer for fast ratiometric measurement of cellular transmembrane potential. J. Phys. Chem. B 110, 13624.

3) Kucherak, O. A. ; Oncul, S. ; Darwich, Z. ; Yushchenko, D. A. ; Arntz, Y. ; Didier, P. ; Mely, Y. ; Klymchenko, A. S. : Switchable Nile Red-Based Probe for Cholesterol and Lipid Order at the Outer Leaflet of Biomembranes. Journal of the American Chemical Society 2010, 132, 4907-4916.

4) Kreder, R. ; Pyrshev, K. A. ; Darwich, Z. ; Kucherak, O. A. ; Mely, Y. ; Klymchenko, A. S. : Solvatochromic Nile Red Probes with FRET Quencher Reveal Lipid Order Heterogeneity in Living and Apoptotic Cells. ACS Chemical Biology 2015, 10, 1435-1442.

5) Niko, Y. ; Didier, P. ; Mely, Y. ; Konishi, G. ; Klymchenko, A. S. : Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes. Scientific Reports 2016, 6, 18870.

6) Darwich, Z. ; Klymchenko, A. S. ; Kucherak, O. A. ; Richert, L. ; Mely, Y. : Detection of apoptosis through the lipid order of the outer plasma membrane leaflet. Biochimica Et Biophysica Acta-Biomembranes 2012, 1818, 3048-3054.

7) Kreder, R. ; Oncul, S. ; Kucherak, O. A. ; Pyrshev, K. A. ; Real, E. ; Mely, Y. ; Klymchenko, A. S. : Blue fluorogenic probes for cell plasma membranes fill the gap in multicolour imaging. Rsc Advances 2015, 5, 22899-22905.

8) Collot, M. ; Kreder, R. ; Tatarets, A. L. ; Patsenker, L. D. ; Mely, Y. ; Klymchenko, A. S. : Bright fluorogenic squaraines with tuned cell entry for selective imaging of plasma membrane vs. endoplasmic reticulum. Chemical Communications 2015, 51, 17136-17139.