Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy

Abstract

Lipid droplets (LDs) are highly dynamic organelles that perform multiple functions, including the regulated storage and release of cholesterol and fatty acids. Information on the molecular composition of individual LDs within their cellular context is crucial in understanding the diverse biological functions of LDs, as well as their involvement in the development of metabolic disorders such as obesity, type II diabetes, and atherosclerosis. Although ensembles of LDs isolated from cells and tissues were analyzed in great detail, quantitative information on the heterogeneity in lipid composition of individual droplets, and possible variations within single lipid droplets, is lacking. Therefore, we used a label-free quantitative method to image lipids within LDs in 3T3-L1 cells. The method combines submicron spatial resolution in three dimensions, using label-free coherent anti-Stokes Raman scattering microscopy, with quantitative analysis based on the maximum entropy method. Our method allows quantitative imaging of the chemistry (level of acyl unsaturation) and physical state (acyl chain order) of individual LDs. Our results reveal variations in lipid composition and physical state between LDs contained in the same cell, and even within a single LD.

FIGURE 1

Illustration of quantitative nature of multiplex CARS. (a) Im{χ(3)} spectra of LA/PA mixtures in CCl₄ in the CC-stretch spectral region. The PA/LA molar ratio is indicated. (b) C = C spectral parameter (I₋₁₆₅₀/I₋₁₄₅₀), as derived from spectra in a, as a function of C = C double-bond concentration. (c and d) Im{χ(3)} spectra in CH-stretch vibrational region of different concentrations of PA in CCl₄, and of mixtures of PA and LA (100 mM total fatty-acid concentration) in CCl₄, respectively. (e) Integrated intensity of CH-stretch Im{χ(3)} spectra of a concentration series of PA in CCl₄ (red triangles) and PA/LA mixtures in CCl₄ (blue squares; 100 mM total fatty -acid concentration).

FIGURE 2

Multiplex CARS spectral imaging. (a) Bright-field image of an adipocyte incubated with a 1:3 mixture (mol/mol) of LA and PA. Marked region was imaged with multiplex CARS microscopy. Based on measured Im{χ(3)} spectra at every pixel, images are derived with contrast based on (d) lipid concentration (integrated intensity), (e) C = C concentration derived from CC-stretch region (I₋₁₆₅₀/I₋₁₄₅₀), and (f) acyl-chain order (I₋₂₈₈₀/I₋₂₈₄₅). (b and c) Representative Im{χ(3)} spectra were recorded at locations indicated (in d) for CC-stretch and CH-stretch spectral regions, respectively. Least-squares fit of a sum of three Lorentzians to the data is also shown (b). Acquisition time was 20 ms for a multiplex CARS spectrum at every pixel position (∼1 min/image).

FIGURE 3

Lipid-droplet composition and packing dependent on incubation medium. Multiplex CARS images are shown for adipocytes incubated with different mixtures of LA and PA. Contrast in first column (a, d, g, and j) is based on lipid concentration (integrated intensity). In second (b, e, h, and k) and third (c, f, i, and l) columns, CC_order (I₋₂₈₈₀/I₋₂₈₄₅) and C = C (I₋₁₆₅₀/I₋₁₄₅₀) parameters are displayed, respectively. (m and n) Values for C = C and CC_order are given, respectively, averaged over ∼20 LDs from at least six different adipocyte cells. For PA fractions ≤50%, LDs show homogeneous unsaturation and acyl-chain order levels. Error bars denote measured variation (1 SD). At PA fractions >50% (m and n, shaded areas), significant heterogeneity between and within LDs is evident, and only individual data points are shown. Because a variety of distribution patterns occurred for the latter incubation conditions, only g, h, i, j, k, and l are exemplary. Transition between homogeneous distributions of unsaturation and order parameters over LDs, and occurrence of heterogeneity within and/or between LDs, are indicated in m and n by shaded regions at elevated PA fractions. Dashed lines indicate reference value obtained for LDs formed in 3T3-L1 cells incubated in medium without added fatty acid (but containing 2% serum; see Methods). Scale bars, 5 μm.

FIGURE 4

Fatty-acid uptake initially results in many small LDs. Multiplex CARS image of LDs in adipocyte after changing incubation medium from pure PA to pure LA. Contrast was based on (a) fatty-acid concentration, and (b) on C = C (I₋₁₆₅₀/I₋₁₄₅₀ intensity ratio). Scale bar, 5 μm.