Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding

Abstract

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

Figure 1. Mean instantaneous D fit for different anti-EGFR Affibody conjugates.

Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

Figure 2. Effect of logD and charge on affibody conjugate mobility.

Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 (A), and logD (B). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R² values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

Figure 3. Plots of distributions of mean instantaneous D fits for affibody-dye conjugates.

Dyes selected to represent high (Alexa 488), moderate (CF 633), low (Alexa 546), and very low (Atto 647N) spot mobility.

Figure 4. Analysis of definitely mobile vs immobile or very slow moving spots.

A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm²/s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm²/s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values <0.1 µm²/s. Line shows linear regression fit to the data, R² value indicating goodness of fit.

Figure 5. Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody.

Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1^st and 3^rd quartile of the distributions.