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.