Mikhail N. Slipchenko,† Thuc T. Le,† Hongtao Chen,‡ and Ji-Xin Cheng*,†,‡

Weldon School of Biomedical Engineering and Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907

Received: March 12, 2009

Cells store excess energy in the form of cytoplasmic lipid droplets. At present, it is unclear how different types of fatty acids contribute to the formation of lipid droplets. We describe a compound Raman microscope capable of both high-speed chemical imaging and quantitative spectral analysis on the same platform. We used a picosecond laser source to perform coherent Raman scattering imaging of a biological sample and confocal Raman spectral analysis at points of interest. The potential of the compound Raman microscope was evaluated on lipid bodies of cultured cells and live animals. Our data indicate that the in vivo fat contains much more unsaturated fatty acids (FAs) than the fat formed via de novo synthesis in 3T3-L1 cells. Furthermore, in vivo analysis of subcutaneous adipocytes and glands revealed a dramatic difference not only in the unsaturation level but also in the thermodynamic state of FAs inside their lipid bodies. Additionally, the compound Raman microscope allows tracking of the cellular uptake of a specific fatty acid and its abundance in nascent cytoplasmic lipid droplets. The high-speed vibrational imaging and spectral analysis capability renders compound Raman microscopy an indispensible analytical tool for the study of lipid-droplet biology.

Introduction

Obesity is an established risk factor for type II diabetes, hypertension, strokes, many types of cancer, atherosclerosis, and other diseases.1,2 A central goal of obesity studies is to understand how cells store excess energy in the form of cytoplasmic lipid droplets (LDs).3 As lipid synthesis and storage pathways are conserved among many organisms, cell model systems derived from simple organisms have been employed to bring insight into the cause of obesity in humans.4 Use of a murine fibroblast-derived 3T3-L1 cell line developed by Green and Kehinde has allowed the transcriptional regulation of fat cell differentiation to be elucidated.4,5 In recent years, a number of lipid-binding proteins have been identified, and their functions in LD formation and mobilization have been characterized.6 Furthermore, a genome-wide RNA interference screen in Drosophila S2 cells revealed a role of phospholipid synthesis in regulating LD size, number, and morphology.7 Nonetheless, significant details on the biology of LDs are still lacking.6,8 Currently, it is not clearly understood how different types of phospholipids or fatty acids contribute to the formation of LDs. Consequently, nutritional intervention in obesity is largely based on restriction of calorie uptake. Effective obesity intervention based on dietary composition is not yet possible because of a lack of understanding of the roles of nutritional ingredients in LD formation.

Until recently, studies of lipid-droplet biology have relied on nonspecific, invasive, or population measurements. Traditionally, intracellular LDs have been visualized based on the fluorescence of lipophilic dyes such as Oil red O (ORO) or Nile red.5,9 The use of ORO, in particular, requires cell fixation, which prevents dynamic studies of LD mobilization and has also been shown to fuse LDs.10 More importantly, because most lipid or fatty acid (FA) molecules have no known specific markers, the fluorescence signals from ORO or Nile red contain no information regarding lipid composition or organization. To analyze the composition of LDs, standard techniques including gas chromatography, liquid chromatography, and mass spectrometry have been employed. Although such analytical techniques are powerful, they provide only population-average information.

Recent advances in vibrational imaging are opening up exciting opportunities for dynamic, noninvasive, and compositional analysis of single LDs. Confocal Raman microscopy allowed visualization of arachidonic acids in LDs inside leukocytes.11 However, long acquisition times on the order of seconds per pixel restrict the use of Raman microscopy to relatively static samples. To increase the vibrational signal level without additional labeling, coherent anti-Stokes Raman scattering (CARS) microscopy has been developed12 and employed to visualize lipid-rich structures at the speed of a few microseconds per pixel or 1 s per frame on a laser-scanning microscope platform.13-15 The large CARS signal is produced by quadratic dependence on the concentration of molecular vibration in the focal volume and by spectral focusing of all of the laser energy into a single Raman band such as the CH2 symmetric stretch mode with picosecond pulse excitation. Such single-frequency CARS microscopy has been employed to monitor LD formation and mobilization in live cells and C. elegans.10,16-18 A drawback of single-frequency CARS microscopy is its lack of spectral information. Multiplex CARS (MCARS) microscopy using broadband pulses has been devised to overcome this shortcoming19,20 and has recently been applied to study the level of fatty acid saturation and the thermodynamic state of LDs in fixed 3T3-L1 cells.21 Compared to Raman microscopy, the vibrational signal in M-CARS is enhanced via mixing with the nonresonant background and can be extracted by the maximum entropy method.21 However, compared to single-frequency CARS, the M-CARS signal is significantly weaker because the excitation energy is spread over a broad

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