Automated quantitative analysis of lipid accumulation and hydrolysis in living macrophages with label-free imaging.
The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R(2) > 0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K(i). Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders.
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, 106, Taiwan.
Wei-Wen
WW
Chen
Chen-Hao
CH
Chien
Chiung-Lin
CL
Wang
Huai-Hsien
HH
Wang
Yuh-Lin
YL
Wang
Shih-Torng
ST
Ding
Tzong-Shyuan
TS
Lee
Ta-Chau
TC
Chang
D000818:Animals; D002460:Cell Line; D006868:Hydrolysis; D050356:Lipid Metabolism; D008055:Lipids / Q000032:analysis*; D008264:Macrophages / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D008853:Microscopy / Q000379:methods*; D013059:Spectrum Analysis, Raman / Q000379:methods*
D008055:Lipids