Label-Free Digital Quantification of Lipid Droplets in Single Cells by Stimulated Raman Microscopy on a Microfluidic Platform

Quantitative characterization of
a single-cell phenotype remains
challenging. We combined a scalable microfluidic array of parallel
cell culture chambers and stimulated Raman scattering (SRS) microscopy
to quantitatively characterize the response of lipid droplet (LD)
formation to free-fatty-acid stimuli with single-LD resolution at
the single-cell level. By enabling the systematic live-cell imaging
with SRS microscopy in a microfluidic device, we were able to quantify
the morphology of over a thousand live cells in 10 different chemical
environments and with 8 replicates for each culture condition, in
a single experiment, and without relying on fluorescent labeling.
We developed an image processing pipeline for cell segmentation and
LD morphology quantification using dual-channel SRS images. This allows
us to construct distributions of the morphological parameters of LDs
in the cellular population and expose the vast phenotypic heterogeneity
among genetically similar cells. Specifically, this approach provides
an analytical tool for quantitatively investigating LD morphology
in live cells in situ. With this high-throughput, high-resolution,
and label-free method, we found that LD growth dynamics showed considerable
cell to cell variation. Lipid accumulation in nonadipocyte cells is
mainly reflected in the increase of LD number, as opposed to an increase
in their size or lipid concentration. Our method allows statistical
single-cell quantification of the LD distribution for further investigation
of lipid metabolism and dynamic behavior, and also extends the possibility
to couple with other “omics” technologies in the future.