Video files: Some images may require stereoscopic glasses / Fat in milk and cream is present as tiny droplets, which are each enveloped in a thin membrane, called the milk fat globule membrane (MFGM). The MFGM can easily be damaged by factors such as pumping the milk and applying other forms of agitation. MFGM damage is believed to reduce processing efficiency and compromise the quality of manufactured products. A comprehensive review of the literature showed that our understanding of changes occurring in the MFGM post secretion of the fat globule by the mammary secretory cell is still rudimentary. Furthermore, it was found that a fundamental understanding of MFGM damage in raw milk is lacking. Hence, this study sought to develop analytical techniques for studying the MFGM. Fluorescent probes were identified that associated with the MFGM (bovine, ovine, human) in one of two ways: either by embedding in the phospholipid bilayer (lipophilic probe) or by binding to carbohydrate moieties of glycosylated chains in the glycocalyx (lectin probes). The use of these probes, in combination with either conventional fluorescence microscopy or confocal laser scanning microscopy, allowed 2-D images and 3-D images of fat globules to be made. Application of water-soluble lipophilic probes and the lectin wheat germ agglutinin (WGA) directly to milk allowed the staining of the MFGM in its native environment. Variable distribution patterns of the probes in the MFGM were observed, which suggests that the MFGM of fat globules in harvested milk is structurally and chemically heterogeneous both within and among globules from the same species and between species, and even among fat globules within the milk of an individual animal. Furthermore, the binding behaviour of WGA to the MFGM of native fat globules (in bovine milk) and washed fat globules (in model systems) following heat treatment implicated β-lactoglobulin, α-lactalbumin, immunoglobulin M and/or the glycosylated proteins Periodic acid Schiff 6/7 in the disappearance of fat globule aggregation upon elevated heat treatment of milk. The results of the current study showed that the use of membrane-specific fluorescent probes, particularly in combination with confocal laser scanning microscopy, has significant potential for providing real time structural and chemical information about the MFGM in matrices such as harvested milk and milk products. In addition to the fluorescence microscopy techniques, development of other techniques was also conducted. Flow cytometry was shown to have significant potential for the quantitative determination of various properties of fat globules and their membranes. Although no suitable sample preparation technique could be developed in this study, atomic force microscopy is believed to have significant potential for studying structural and physical properties of the MFGM. Selective harvesting of individual fat globules was shown to be possible by using a micromanipulator. In future work, this technique is expected to be used in combination with fluorescence microscopy, or atomic force microscopy. The present study has shown that the development and application of novel analytical techniques has advanced, and in the future will further advance, understanding of the MFGM.