Lipid droplet quantification based on iterative image processing.

Lipid droplets (LDs) are ubiquitous and highly dynamic subcellular organelles required for the storage of neutral lipids. LD number and size distribution are key parameters affected not only by nutrient supply but also by lipotoxic stress and metabolic regulation. Current methods for LD quantification lack general applicability and are either based on time consuming manual evaluation or show limitations if LDs are high in numbers or closely clustered. Here, we present an ImageJ-based approach for the detection and quantification of LDs stained by neutral lipid dyes in images acquired by conventional wide-field fluorescence microscopy. The method features an adjustable preprocessing procedure that resolves LD clusters. LD identification is based on their circular edges and central fluorescence intensity maxima. Adaptation to different cell types is mediated by a set of interactive parameters. Validation was done for three different cell lines using manual evaluation of LD numbers and volume measurement by 3D rendering of confocal datasets. In an application example, we show that overexpression of the acyl-CoA synthetase, FATP4/ACSVL5, in oleate-treated COS7 cells increased the size of LDs but not their number.