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. 2022 Mar 18;12(3):468.
doi: 10.3390/biom12030468.

Impact of Molecule Concentration, Diffusion Rates and Surface Passivation on Single-Molecule Fluorescence Studies in Solution

Olessya Yukhnovets  1 , Henning Höfig  1 , Nuno Bustorff  2 , Alexandros Katranidis  2 , Jörg Fitter  1   2
Affiliations

Impact of Molecule Concentration, Diffusion Rates and Surface Passivation on Single-Molecule Fluorescence Studies in Solution

Olessya Yukhnovets et al. Biomolecules. .

Erratum in

Abstract

For single-molecule studies in solution, very small concentrations of dye-labelled molecules are employed in order to achieve single-molecule sensitivity. In typical studies with confocal microscopes, often concentrations in the pico-molar regime are required. For various applications that make use of single-molecule Förster resonance energy transfer (smFRET) or two-color coincidence detection (TCCD), the molecule concentration must be set explicitly to targeted values and furthermore needs to be stable over a period of several hours. As a consequence, specific demands must be imposed on the surface passivation of the cover slides during the measurements. The aim of having only one molecule in the detection volume at the time is not only affected by the absolute molecule concentration, but also by the rate of diffusion. Therefore, we discuss approaches to control and to measure absolute molecule concentrations. Furthermore, we introduce an approach to calculate the probability of chance coincidence events and demonstrate that measurements with challenging smFRET samples require a strict limit of maximal sample concentrations in order to produce meaningful results.

Keywords: burst analysis; chance coincidence probability; confocal fluorescence microscopy; single-molecule Förster resonance energy transfer; two-color coincidence detection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
BTCCD results of double-labelled dsDNA samples as obtained from measurements at standard buffer conditions (only PEGylated surfaces, see Section 2.1 and Section 2.6). (a) Coincidence fractions, fRB (blue) and fBR (red), are shown as a function of the brightness-gating parameter nbr for consecutive 10 min time intervals after the measurements were started. (b) On the basis of the data shown in (a), the time course of the dye concentration in solution was provided in terms of “total number of bursts” as obtained in the respective time interval (see the left y-axis) and in the molar concentration (see the right y-axis). (c) On the basis of the data shown in (a), the time course of the individual coincidence fractions fRB (blue) and fBR (red) are shown.
Figure 2
Figure 2
BTCCD results of double-labelled dsDNA samples. (a,b) For data which were obtained from measurements in buffers containing 0.005% Tween20, the same types of graphs are shown as in Figure 1b,c, respectively.
Figure 3
Figure 3
Results as obtained from smFRET measurements with PGK at 0.65 M GuHCl denaturant concentration. (a) The number of detected bursts and the related molar protein concentration was given during the total measuring time (60 min) for time intervals of 10 min. (b,c) The corresponding smFRET transfer efficiency histograms are shown for the first 30 min (b) and for the last 30 min (c). In both histograms, the unfolded state population (at <E1> ≈ 0.2) and the folded state population (at <E2> ≈ 0.75) were within the limits of error of the same statistical weights P1 ≈ 0.45 and P2 ≈ 0.55, respectively (area under the respective fitted Gaussian peaks).
Figure 4
Figure 4
Measured (solid lines) and calculated (dashed lines) fRB values obtained from samples with a pair of two distinct individually and freely diffusing molecules, one labelled red (Alexa647/Atto647N/Cy5) and the other labelled blue (Alexa488). The respective molecule concentrations are given for the blue-labelled species (including the parameter N, the corresponding average number of molecules in the detection volume at the same time). The concentration of the red-labelled species (not given explicitly) was in the same regime as the blue-labelled species. The presented examples exhibited a clear variation in the relative sizes, and thereby in the corresponding burst duration times, τd, of the respective molecules in the mixed samples: (a) similar size of red- and blue-labelled molecules, (b) larger red-labelled molecule versus smaller blue-labelled molecule (i.e., only Alexa488), and (c) very large red-labelled molecule versus small blue-labelled molecule.
Figure 4
Figure 4
Measured (solid lines) and calculated (dashed lines) fRB values obtained from samples with a pair of two distinct individually and freely diffusing molecules, one labelled red (Alexa647/Atto647N/Cy5) and the other labelled blue (Alexa488). The respective molecule concentrations are given for the blue-labelled species (including the parameter N, the corresponding average number of molecules in the detection volume at the same time). The concentration of the red-labelled species (not given explicitly) was in the same regime as the blue-labelled species. The presented examples exhibited a clear variation in the relative sizes, and thereby in the corresponding burst duration times, τd, of the respective molecules in the mixed samples: (a) similar size of red- and blue-labelled molecules, (b) larger red-labelled molecule versus smaller blue-labelled molecule (i.e., only Alexa488), and (c) very large red-labelled molecule versus small blue-labelled molecule.
Figure 5
Figure 5
Here, stoichiometry versus FRET efficiency plots are shown for RNC samples as measured with different molecule or dye concentrations. (a) At higher concentrations, the corresponding FRET efficiency histogram (upper panel) showed only peaks at <E> ≈ 1 and <E> ≈ 0, which can be considered as artifacts. (b) At lower concentrations, the corresponding efficiency histogram exhibited a meaningful FRET population at <E> ≈ 0.55, that represented the apo-CaM state. The color code gives the number of burst for each point in the S vs. E plane.
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