Figure 1.

Thresholding-based identification of lipid droplets (LDs) in HeLa cells with varying LD density. (A) Object identification strategy. A median-filtered background (neighborhood size, 128 pixels) was subtracted from the original image. The image was then thresholded, with pixels measuring <15% of the maximum pixel intensity set to zero. All contiguous sets of pixels were identified, and pixels in each cluster with a value less than 50% of the maximum pixel value for that cluster were set to zero. (B) HeLa cells incubated with 350 mM oleic acid for 24 hr and then stained with Nile Red. The field shown was selected to show an example with well-separated LDs. (C) HeLa cells incubated with 2 mM oleic acid for 24 hr and stained with Nile Red to generate large areas of near-confluent LDs. (D) HeLa cells incubated and stained as in (A), but field chosen to illustrate some degree of LD clustering under moderate labeling conditions. All source images (left; B, C, D) were prepared by projection and median filter background correction of z-stacks following the procedure detailed in the Materials & Methods. Scale, 5 µm.