DiI, DiO, DiD and DiR dyes are a family of lipophilic fluorescent stains for labeling membranes and other hydrophobic structures. The fluorescence of these environment-sensitive dyes is greatly enhanced when incorporated into membranes or bound to lipophilic biomolecules such as proteins although they are weakly fluorescent in water. They have high extinction coefficients, polarity-dependent fluorescence and short excited-state lifetimes. Once applied to cells, these dyes diffuse laterally within the cellular plasma membranes, resulting in even staining of the entire cell at their optimal concentrations. The distinct fluorescence colors of DiI (orange fluorescence), DiO (green fluorescence), DiD (red fluorescence) and DiR (deep red fluorescent) provide a convenient tool for multicolor imaging and flow cytometric analysis of live cells. DiO and DiI can be used with standard FITC and TRITC filters respectively. Among them DiD is well excited by the 633 nm He–Ne laser, and has much longer excitation and emission wavelengths than those of DiI, providing a valuable alternative for labeling cells and tissues that have significant intrinsic fluorescence. DiR might be useful for in vivo imaging or tracing due to the effective transmission of infrared light through cells and tissues and low level of autofluorescence in the infrared range. This particular DiI derivative is an amphiphilic molecule that contains both hydrophilic and lipophilic moiety. It has stronger fluorescence in water than the highly lipophilic DiI analogs.
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