Fluorescent labeling allows for imaging and tracking of vesicles down to single-particle level. Among several options to introduce fluorescence, staining of lipid membranes with lipophilic dyes provides a straightforward approach without interfering with vesicle content. However, incorporating lipophilic molecules into vesicle membranes in an aqueous solution is generally not efficient because of their low water solubility.
Researchers at the Pohang University of Science and Technology describe a simple, fast (<30 min), and highly effective procedure for fluorescent labeling of vesicles including natural extracellular vesicles. By adjusting the ionic strength of the staining buffer with NaCl, the aggregation status of DiI, a representative lipophilic tracer, can be controlled reversibly. Using cell-derived vesicles as a model system, the researchers show that dispersion of DiI under low-salt condition improved its incorporation into vesicles by a factor of 290. In addition, increasing NaCl concentration after labeling induced free dye molecules to form aggregates, which can be filtered and thus effectively removed without ultracentrifugation. The researchers consistently observed 6- to 85-fold increases in the labeled vesicle count across different types of dyes and vesicles. The method is expected to reduce the concern about off-target labeling resulting from the use of high concentrations of dyes.
Fluorescent labeling of vesicles by salt-change method
(A, B) Schematics of direct staining (A) and salt-change (B) method for the labeling of cell-derived vesicles (CDVs) with DiI in an aqueous buffer. In the salt-change method, staining was performed in a low-salt buffer, then [NaCl] was increased to 150 mM to promote aggregation of DiI, and the aggregates were removed by filtration. (C) Schematic of single-vesicle imaging of DiI-labeled CDVs by TIRF microscopy. (D) Representative fluorescent images of DiI-labeled vesicles. Scale, 10 μm. (E) Numbers of DiI-labeled vesicles obtained by direct staining and salt-change method observed in TIRF images. Error bars, mean ± s.d. of n = 63 images. (F) Distributions of fluorescence intensity for the labeled vesicles. Inset, close-up view of the same curves; n = 56 (direct staining), n = 4671 (salt change) spots.