Microstructure and biomolecules mobility of human milk fat globules by fluorescence recovery after photobleaching with confocal scanning laser microscope

Abstract

This work demonstrates the use of fluorescence recovery after photobleaching (FRAP) for direct measurement of the microstructure and biomolecules mobility of human milk fat globules (HMFGs). In particular, five fluorescent probes (three types), Nile Red, Rh-DOPE, NBD-PC, NBD-SM, and Nile Blue, were evaluated in a Confocal scanning laser microscope combined with FRAP for the detection of the molecular diffusion of neutral lipids, polar lipids and membrane proteins in HMFGs. Mobile fraction (Mf), half-life recovery time (t1/2) and diffusion coefficient (K) were calculated from the FRAP recovery curve of five fluorescent molecules. The Mf of fluorescent probes labeled neutral lipids, polar lipids, membrane proteins, phosphatidylcholine, sphingomyelin, and on HMFGs were 63%, 18%, 45%, 28% and 5%, respectively. The recovery times of fluorescent molecules labeled with membrane biomolecules were longer as the size of the milk fat globules decreased and the photobleaching area increased. This method was applied to the HMFG membrane cytoplasmic remnants (CR) region showing a similar trend and much higher fluorescence intensity to the membrane region, which suggests that CR might contain multiple layers of membrane lipids.