Two-Color Visualization of Cholesterol Fluctuation in Plasma Membranes by Spatial Distribution-Controllable Single Fluorescent Probes.

Visualizing cholesterol (CL) fluctuation in plasma membranes is a crucially important yet challenging task in cell biology. Here, we proposed a new imaging strategy based on permeability changes of plasma membranes triggered by different CL contents to result in controllable spatial distribution of single fluorescent probes (SF-probes) in subcellular organelles. Three spatial distribution-controllable SF-probes (PMM-Me, PMM-Et, and PMM-Bu) for imaging CL fluctuation in plasma membranes were rationally developed. These SF-probes target plasma membranes and mitochondria at normal CL levels, while they display solely staining in plasma membranes and mitochondria at increased and decreased CL levels, respectively. These polarity-sensitive probes also show distinct emission colors with fluorescence peaks of 575 and 620 nm in plasma membranes and mitochondria, respectively. Thus, the CL fluctuation in plasma membranes can be clearly visualized by means of the spatially distributed and two-color emissive SF-probes.