Greater progress in understanding the microstructure of the milk fat globule (membrane) [MFG(M)] could be made if microscopy techniques that do not require the isolation of the fat globules from their native environment prior to microscopy imaging were available. In this study, fluorescence microscopy techniques were developed for studying the fat globules in milk in real time. Screening of a range of commercially available fluorescent probes showed that some probes diffused heterogeneously into the core of the fat globules, whereas other probes located preferentially in the MFGM. Using confocal laser scanning microscopy, the partially water-soluble probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid, in particular, has significant potential for providing structural information about the MFGM in milk. Such information is important for obtaining a fundamental understanding of the effects of milk handling and treatment on the MFGM.