Spatial localization of charged molecules by salt ions in oil-confined water microdroplets

Abstract

Cells contain more than 100 mM salt ions that are typically confined to dimensions of 5 to 10 micrometers by a hydrophobic cellular membrane. We found that in aqueous microdroplets having the same size as cells and that are confined in hydrocarbon oil, negatively charged molecules were distributed rather uniformly over the interior of the microdroplet, whereas positively charged molecules were localized at and near the surface. However, the addition of salt (NaCl) to the microdroplet caused all charged molecules to be localized near the oil-water interface. This salt-induced relocalization required less salt concentration in microdroplets compared to bulk water. Moreover, the localization became more prominent as the size of the microdroplet was reduced. The relocatization also critically depended on the type of oil. Our results imply that salt ions and different hydrophobic interfaces together may govern the local distribution of charged biomolecules in confined intracellular environments.

Fig. 1. Spatial distributions of the fluorescent dyes.

(A) MitoTracker Red FM (MitoTracker), (B) Alexa Fluor 647 (AF647), (C) Alexa Fluor 555 (AF555), (D) 10-bp DNA-AF647, and (E) 30-bp DNA-AF647 in aqueous microdroplets suspended in hydrocarbon immersion oil. The chemical structures of the fluorescent dyes with their charges are provided in the left panels, and fluorescence and bright-field images in the absence of salt ions and in the presence of 100 mM NaCl are presented in the middle and right panels, respectively (scale bars, 5 μm).

Fig. 2. Spatial distribution of AF647 fluorescent dye at two different cross-sectional planes of the microdroplet (48 μm in diameter).

(A) Schematic of imaging conditions. (B and C) Fluorescence images acquired at the 15 μm below (left) and 15 μm above (right) the equator of the microdroplet. (D) Distributions of AF647 dyes at the two different imaging planes of the microdroplet. a.u., arbitrary units.

Fig. 3. Effect of different NaCl concentrations on the localization of negatively charged fluorescent dye molecules and DNA molecules tagged with AF647.

The fluorescence/bright-field overlay images of aqueous microdroplets containing AF647 (A), AF555 (B), 10-bp DNA-AF647 (C), and 30-bp DNA-AF647 (D) with the indicated NaCl concentrations (scale bars, 5 μm). The normalized spatial distributions of each dye under various NaCl concentrations are plotted in the bottom panel. Because different sizes of microdroplets were analyzed, the distance from the center to the boundary of each microdroplet was normalized to one and divided into 10 segments. Fluorescence intensity was averaged in each segment and plotted as a function of the relative distance from the center to the periphery of microdroplets. Error bars represent 1 SD from five independent measurements.

Fig. 4. Different spatial distribution of fluorescent dye in microdroplets and bulk solution.

(A) Spatial distribution of the fluorescent dye, AF647, in aqueous microdroplets larger than 7 μm in diameter in the absence (black lines) and the presence of 100 mM NaCl (red lines). The microdroplets were interfaced to the immersion oil. The relative fluorescence intensities were plotted against the distances from the droplet center to the interface. (B) Spatial distribution of fluorescent dye, AF647, in bulk water containing different salt concentrations from 0 to 100 mM NaCl, which is in contact with immersion oil. Each fluorescence image was recorded on the parallel section above the cover glass by 4.8 μm to avoid the interfering fluorescence signals on the cover glass surface (scale bars, 5 μm). The fluorescence intensities at the interfacial region were plotted against the distance from the interior aqueous part to the oil part, and the contact boundary was marked by the dashed magenta line.

Fig. 5. Spatial localization of dyes for various oil-water interfaces.

The changes in the molecular localization of AF647 under various NaCl concentrations when interfacing with different hydrophobic oils; triolein (A and B), olive oil (C and D), and silicone oil (E and F). Overlay of fluorescence and bright-field images and their distribution profiles of AF647 in microdroplets surrounded by each oil (A, C, and E) and fluorescence images near the interface between bulk oil and bulk water (B, D, and F) containing different concentrations of NaCl (scale bars, 5 μm). Error bars represent 1 SD from five independent measurements.

Fig. 6. Effect of different types of cations on the peripheral localization of AF647 in microdroplets.

The “Periphery” region is defined as the region where the radius is more than 70% of the whole radius of each droplet. The average fluorescence intensities in the Periphery are compared with those in the whole droplet regions; the value 1.0 reflects that the fluorescent dyes are evenly distributed in the microdroplet including the periphery. Error bars represent 1 SD from five independent measurements.