Cytoplasmic granules in fibroblasts, visualized without stains, or labelled with
Nile red,
Filipin, or anti-LAMP-1 (
lysosome-associated membrane protein 1), were imaged using the real-time microscope (RTM). New advances in light microscope technology were applied to detect cytoplasmic granules (RTM-visible granules) and characterize them by imaging contrast, size, shape, cellular distribution, composition, motion dynamics and quantity. Appearing as solid spheroids or ring structures, the majority of the RTM-visible granules contained
Nile-red labelled neutral
lipids. A smaller subpopulation, appearing dimmer, with less imaging contrast, contained
Filipin-labelled free
cholesterol. Most
lipid storage granules have a diameter ranging from 0.3 mum to 0.6 mum, with a small population measuring up to 1 mum. They typically clustered in the perinuclear region and displayed relatively small oscillatory motion. Immunofluorescence based on LAMP-1 labelling highlighted granular structures that were distinct and separate from RTM-visible granules and other structures in the light modality of the microscope. RTM-visible granules were associated with disease phenotypes that have increased cellular neutral
lipid stores corresponding to the
Nile red-labelled droplets (e.g. triacylglycerides,
cholesterol esters). As predicted, the fibroblast strains with a defect resulting in
Wolman disease, when compared to control samples, consistently had RTM-visible granules, higher in imaging contrast and with larger diameters, that were labelled with
Nile red, and also an increased frequency of
Filipin-
cholesterol complexes. By comparison, in fibroblasts where the
lipid storage is less evident (Gaucher and Farber diseases) or from GM(1)
gangliosidosis, where the primary storage substances are
oligosaccharides, fewer and smaller RTM-visible granules were observed. In some cases, changes in contrast and morphology in the unstained cytoplasmic compartments were more evident than in the labelled structures. In summary, applying the RTM imaging system to fibroblasts enables differences between the various disease types to be seen and, in specific examples, a unique phenotype can be readily discerned.