Quantitative Spatiotemporal Chemical Profiling of Individual Lipid Droplets by Hyperspectral CARS Microscopy in Living Human Adipose-Derived Stem Cells

Abstract

There is increasing evidence showing that cytosolic lipid droplets, present in all eukaryotic cells, play a key role in many cellular functions. Yet their composition at the individual droplet level and how it evolves over time in living cells is essentially unknown due to the lack of suitable quantitative nondestructive measurement techniques. In this work, we demonstrate the ability of label-free hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy, together with a quantitative image analysis algorithm developed by us, to quantify the lipid type and content in vol/vol concentration units of individual lipid droplets in living human adipose-derived stem cells during differentiation over 9 days in media supplemented with different fatty acids. Specifically, we investigated the addition of the polyunsaturated linoleic and alpha-linolenic fatty acids into the normal differentiation medium (mostly containing monounsaturated fatty acids). We observe a heterogeneous uptake which is droplet-size dependent, time dependent, and lipid dependent. Cells grown in linoleic-acid-supplemented medium show the largest distribution of lipid content across different droplets at all times during differentiation. When analyzing the average lipid content, we find that adding linoleic or alpha-linolenic fatty acids at day 0 results in uptake of the new lipid components with an exponential time constant of 22 ± 2 h. Conversely, switching lipids at day 3 results in an exponential time constant of 60 ± 5 h. These are unprecedented findings, exemplifying that the quantitative imaging method demonstrated here could open a radically new way of studying and understanding cytosolic lipid droplets in living cells.

The why, when and how of lipid droplet diversity

“…2H). For example, coherent anti-stokes Raman scattering (CARS) microscopy has revealed that lipid droplets within human adipose-derived stem cells and microalgae have different lipid constituents (Cavonius et al, 2015;Di Napoli et al, 2016). Furthermore, in McArh7777 rat liver cells, lipid droplets are either enriched in TAG or cholesterol esters (Hsieh et al, 2012).…”

Broadband Coherent Raman Scattering Microscopy

“…After that both pulses propagated through another SF57 glass block before entering the microscope. This configuration was later adopted in many experiments . The major benefit of using a single sub‐10‐fs Ti:sapphire laser over a PCF pumped by Ti:sapphire is the flat and stable spectral phase and high output power.…”

Molecular imaging with CARS micro-spectroscopy

“…As shown in Figure 2, most of the peaks in the fingerprint region (there are roughly 50 of them) are intrinsically quite weak, whereas the roughly five peaks of the CH stretch region (2800 to 3100 cm −1 ) are much stronger. For this reason, coherent Raman imaging studies (even spectroscopic CRI) have focused primarily on lipids and other species that have strong signals in the CH stretch region of the spectrum 39–41 .…”