Automated quantitative analysis of lipid accumulation and hydrolysis in living macrophages with label-free imaging

Abstract

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.

Quantitative imaging of lipid droplets in single cells

“…derived a formula to calibrate CARS intensity to accurately report the number of lipid molecules in the scattering volume. 71 In this study, they developed an automated image analysis algorithm for quantification of lipid content in single cells. Rinia et al adopted another strategy where they implemented spectral-analysis tools in conjunction with multiplex CARS for retrieval of spontaneous Raman-like spectra which is linear with the number of vibrating molecules.…”

Real‐time Raman and SRS imaging of living human macrophages reveals cell‐to‐cell heterogeneity and dynamics of lipid uptake

“…Hence, CRS has been employed to visualize the distribution of lipids and lipid droplets in organisms and cells . In regards to atherosclerotic progression on a cellular level, in particular the lipid accumulation and metabolism in monocytes and macrophages have been studied . Biomedical applications with the focus on CRS‐based lipid imaging have been extensively reviewed .…”

Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging

“…The quantitative analysis of lipid content in oocytes was modified from our previous image analysis method 50 51 . CARS images of oocytes were taken at 2845 cm −1 and analyzed by image J software.…”