Enhanced fluorogenic plasma membrane probe

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Mechanisms and characteristics of enhanced fluorogenic plasma membrane probe

What is the plasma membrane, and why do investigators want to visualize it with fluorescent microscopy?

The plasma membrane (PM) of eukaryotic cells is comprised of a lipid bilayer that not only functions as a continuous barrier to separate the intracellular and extracellular environments, but it also plays critical roles in signal transduction, cell recognition, substance transport, mechanosensing, biological scaffolding, and several other functions^{(reviewed in 1-4)}.

Effectively visualizing the PM is important for cell segmentation, but it is also useful to characterize specific functions of the PM and its impact on other cellular organelles, structures, and processes^{(reviewed in 5)}. Fluorescent microscopy has emerged as a critical approach for studying the PM’s dynamics and morphology, and recent advancements in PM fluorescent probes like MemGlow allow investigators to specifically and fluorogenically label the PM in cells^{6, 7}, while producing superior results compared to older generation membrane probes like WGA, DiD, and PKH⁸.

Specialized probes such as Flipper-TR⁹ (PM membrane tension), NR4A¹⁰ (solvatochromic SMLM), and PKmem¹¹ (gentle, ultralong-term PM imaging) have also been developed, allowing for more specialized investigation of the PM.

What are eFM probes, and how do they add to the current toolbox of PM probes?

Original FM dyes, named after Fei Mao and developed by Betz et. al.¹², were routinely used for membrane labeling, and one of the most popular derivatives, FM1-43, was widely used to label the PM in plants. However, its suboptimal properties produces inefficient PM labeling in many mammalian cell models¹³ (see Figure 1). Its utility is also limited due to its rapid internalization by endocytosis as well as its broad absorption and emission spectra, making it unsuitable for multi-color imaging.

Importantly, the FM1-43 dye has beneficial optical properties, such as its photostability and large Stokes shifts, which may be beneficial for STED super-resolution imaging. Thus, the group that developed the groundbreaking MemGlow probes performed rational chemical modifications (see Figure 2) to neutralize the FM1-43’s cationic nature, alter its absorption spectrum, and improve its PM specificity through the introduction of two amphiphilic moieties to create enhanced FM (eFM) probes that retain the beneficial characteristics of the parental FM1-43 probe while overcoming its drawbacks¹³.

The outcome is the development of two eFM™ probes (eFM488 and eFM555) that exhibit reduced crosstalk, higher brightness, improved photostability (see Figure 3), reduced cytotoxicity, and greater retention within the PM¹³.

The eFM488 probe has a 4-fold reduction in absorption at 530 and 560 nm, enabling multiplexing with probes in the far-red channel. Of note, the eFM488 probe was validated for use in STED microscopy.

The eFM555 received an additional modification that red-shifted both its absorbance and emission spectra, which allows for multi-color imaging with fluorophores in the green channel. Due to eFM555's greater hydrophobicity, it remains more aggregated in PBS, similar to MemGlow, and produces a higher fluorescent enhancement than FM1-43 (53-fold vs 31-fold).

Both eFM probes are effective at labeling mammalian cells (see Figure 1) while retaining their ability to stain plant cells and tissue, a clear improvement over the FM1-43 parental probe. These eFM probes were validated with multiple microscopy techniques and were tested in multiple experimental models¹³.

These probes are great for end users who need a PM-specific probe with large Stoke shifts and high photostability for confocal and STED imaging, using live or fixed mammalian or plant cells.

Sanity Image — Figure 1: Laser scanning confocal imaging of live cells from four different cell lines stained with 1 μMol FM1-43 (A, B, C, D) and SP-468 (E, F, G, H). Image provided courtesy of Mayeul Collot et al. 2020.

For more information on Cytoskeleton's selection of eFM probes, click below

eFM488 Enhanced Fluorogenic Membrane Probe (Cat # MG15)

eFM560 Enhanced Fluorogenic Membrane Probe (Cat # MG16)

References

1. Zhang, T., W. Hu, and W. Chen, Plasma Membrane Integrates Biophysical and Biochemical Regulation to Trigger Immune Receptor Functions. Front Immunol, 2021. 12: p. 613185.

2. Horn, A. and J.K. Jaiswal, Structural and signaling role of lipids in plasma membrane repair. Curr Top Membr, 2019. 84: p. 67-98.

3. Fliegel, L., Structure and Function of Membrane Proteins. Int J Mol Sci, 2023. 24(9).

4. Mukhopadhyay, U., et al., The Plasma Membrane and Mechanoregulation in Cells. ACS Omega, 2024. 9(20): p. 21780-21797.

5. Collot, M., S. Pfister, and A.S. Klymchenko, Advanced functional fluorescent probes for cell plasma membranes. Curr Opin Chem Biol, 2022. 69: p. 102161.

6. Collot, M., et al., MemBright: A Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience. Cell Chem Biol, 2019. 26(4): p. 600-614 e7.

7. Breton, V., et al., Molecular mapping of neuronal architecture using STORM microscopy and new fluorescent probes for SMLM imaging. Neurophotonics, 2024. 11(1): p. 014414.

8. Hyenne, V., et al., Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo. Dev Cell, 2019. 48(4): p. 554-572 e7.

9. Colom, A., et al., A fluorescent membrane tension probe. Nat Chem, 2018. 10(11): p. 1118-1125.

10. Danylchuk, D.I., et al., Switchable Solvatochromic Probes for Live-Cell Super-resolution Imaging of Plasma Membrane Organization. Angew Chem Int Ed Engl, 2019. 58(42): p. 14920-14924.

11. Ling, J., et al., A gentle palette of plasma membrane dyes. Proc Natl Acad Sci U S A, 2025. 122(29): p. e2504879122.

12. Betz, W.J., F. Mao, and G.S. Bewick, Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J Neurosci, 1992. 12(2): p. 363-75.

13. Collot, M., et al., Molecular Tuning of Styryl Dyes Leads to Versatile and Efficient Plasma Membrane Probes for Cell and Tissue Imaging. Bioconjug Chem, 2020. 31(3): p. 875-883.