Chemical imaging of lipid droplets in muscle tissues using hyperspectral coherent Raman microscopy
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The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.
KeywordsLipid droplet Microscopy Chemical imaging Raman spectroscopy Multivariate analysis Hyperspectral
This study was financially supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organization for the Advancement of Research) (M.B., G.R.), the NanoNextNL, a micro and nanotechnology consortium of the Government of the Netherlands and 130 partners (N.B., M.B., and M.K.C.L.), and a Marie Curie Foundation grant #CIG322284 (S.H.P). Ma.B. was financially supported by NUTRIM and the Graduate School VLAG. A Vici (Grant 918.96.618) grant for innovative research from the Netherlands Organization for Scientific Research supports the work of P.S.
G.R. and N.B. performed CARS experiments. P.L. and G.H. helped construct the experimental system. GR and GE did the multivariate analysis using HCA and PCA. A.G., Ma.B., and N.B. performed the fluorescence imaging. N.B., Ma.B., M.K.C.H., and P.S. provided samples. G.R., N.B., M.B., and S.H.P. wrote the paper. M.B. and S.H.P. supervised the research. All authors contributed to discussion of the results and revision of the paper. The authors wish to thank Dr. E. Cánovas and Dr. W. Rock for stimulating discussions and technical support.
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