Chemical imaging of lipid droplets in muscle tissues using hyperspectral coherent Raman microscopy

Abstract

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.

Biological imaging with coherent Raman scattering microscopy: a tutorial

“…110 Note that the differences in the spectra are subtle, yet are sufficient for a reliable mapping of functional units in the tissue. These are the types of applications where the imaging properties of CRS are in full effect: label-free chemical contrast, fast image acquisition, and (sub)micrometer resolution.…”

In Situ and In Vivo Molecular Analysis by Coherent Raman Scattering Microscopy

“…Hyperspectral CARS was used to study lipid storage in muscle tissue (125) and identify cholesterol crystals from condensed aliphatic lipids in atherosclerotic plaques in mouse model (126). Suhalim et al (26) showed that the structure of crystalized cholesterol in plaques can be analyzed by integrating SRS microscopy with second harmonic generation modality (Figure 4 d ); Wang et al (74) discriminated cholesterol crystals from lipids in the pig model, and further quantified the cholesterol ester level and the degree of unsaturation in lipid droplets, by hyperspectral SRS.…”

Label-free imaging and quantitative chemical analysis of Alzheimer’s disease brain samples with multimodal multiphoton nonlinear optical microspectroscopy

“…9,10 In particular, the three techniques of coherent antiStokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second-harmonic generation (SHG) microscopy are most widely employed for biomedical imaging. [11][12][13] Because each modality has different contrast mechanisms and images different biomolecular components, combining those multiphoton microscopies in one platform would make it possible to observe the proportions of various biomolecules.…”