Choose your label wisely: water-soluble fluorophores often interact with lipid bilayers

Abstract

Water-soluble organic fluorophores are widely used as labels in biological systems. However, in many cases these fluorophores can interact strongly with lipid bilayers, influencing the interaction of the target with the bilayer and/or leading to misleading fluorescent signals. Here, we quantify the interaction of 32 common water-soluble dyes with model lipid bilayers to serve as an additional criterion when selecting a dye label.

Figure 1. Example raw fluorescence spectra of three dyes.

The dyes are representative of low (Alexa 647-SE), moderate (Alexa 594-M), and high (Atto 647N-M) MIF values. Fl_{in_raw} is the raw fluorescence spectrum of the vesicle solution inside the dialysis cassette, collected and prepared as described above. Fl_{out_raw} is the raw fluorescence spectrum of the solution outside the dialysis cassette. MIF values are the corrected MIF values averaged across three separate measurements, as reported in Table 1. Emission fluorescence is in arbitrary units.

Figure 2. Bar graph of membrane interaction factors (MIF, Table 1 ), sorted by excitation maximum.

Dyes below the bottom dashed line (MIF<0.1) exhibit little to no association with Egg PC lipid bilayers, while dyes above the second dashed line (MIF>1) strongly associate with membranes. Note that the y-axis changes substantially at MIF>1.1. Each data point represents the average of three independent measurements, ± propagated error of the standard deviation.

Figure 3. Correlation of MIF values with calculated log D.

Calculated log D values are given in Table 1. (A) The MIF value shows moderate correlation with calculated log D values, based on the unhydrolyzed dye structures. (B) Zoom-in on dyes with MIF values below 1. Dyes with a MIF below the bottom dashed line (MIF<0.1) show little association with membranes, and dyes above the top dashed line (MIF>1) show appreciable interaction with lipid bilayers. MIF values shown here are the MIF_corr values given in Table 1.

Figure 4. MIF_{corr_neg} values for a subset of dyes interacting with negatively charged 9∶1 Egg PC:DOPS vesicles.

For comparison, the corrected MIF values of the dyes with pure Egg PC (MIF_corr, solid bars) are shown to the left of the MIF values with DOPS (MIF_{corr_neg,} hashed bars). The MIF values in both lipid compositions, calculated as described in Equation 3, are displayed above each bar, and are given in Table 2. Dyes below the bottom dashed line (MIF<0.1) exhibit little to no association with Egg PC lipid bilayers, while dyes above the second dashed line (MIF>1) strongly associate with membranes. Note that the y-axis changes substantially at MIF>1.1. Each data point represents the average of three independent measurements, ± propagated error of the standard deviation. The difference between MIF_{corr_neg} and MIF_corr for all dyes shown is statistically significant at the p<0.05 level, as determined by a two-sample Kolmogrov-Smirnov test.

Figure 5. Observation of fluorophores interacting with lipid bilayers by fluorescence microscopy.

After incubating a dye solution with a supported lipid bilayer and rinsing, any remaining fluorescence was imaged. The Alexa 647-M and Abberior STAR 635P azide images are set to the same contrast, while the Alexa 633-M sample was appreciably brighter.