Raman Imaging Providing Insights into Chemical Composition of Lipid Droplets of Different Size and Origin: In Hepatocytes and Endothelium Katarzyna Majzner, †,‡ Kamila Kochan, †,‡ Neli Kachamakova-Trojanowska, † Edyta Maslak, † Stefan Chlopicki, †,§ and Malgorzata Baranska* ,†,‡ † Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, Krakow, 30-348, Poland ‡ Faculty of Chemistry, Jagiellonian University, Ingardena 3, Krakow, 30-060, Poland § Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University, Grzegorzecka 16, Krakow, 31-531, Poland * S Supporting Information ABSTRACT: In this work, 3D linear Raman spectroscopy was used to study lipid droplets (LDs) ex vivo in liver tissue and also in vitro in a single endothelial cell. Spectroscopic measurements combined with fluorescence microscopy and/or histochemical staining gave complex chemical information about LD composition and enabled detailed investigations of the changes occurring in various pathological states. Lipid analysis in fatty liver tissue was performed using a dietary mouse model of liver steatosis, induced by a high fat diet (HFD). HFD is characterized by a high percentage of calories from saturated fat (60%) and reflects closely the detrimental effects of dietary habits responsible for increased morbidity due to obesity and its complications in well-developed Western societies. Such diets lead to obesity, hyperlipidemia, insulin resistance, and steatosis that may also be linked to endothelial dysfunction. In the present work, Raman spectroscopy was applied to characterized chemical composition of lipid droplets in hepatocytes from mice fed HFD and in the endothelium treated with exogenous unsaturated free fatty acid (arachidonic acid). The results demonstrate the usefulness of Raman spectroscopy to characterize intracellular lipid distribution in 2D and 3D images and can be used to determine the degree of saturation. Raman spectroscopy shows the potential to be a valuable tool for studying the role of LDs in physiology and pathology. The method is generally applicable for the determination of LDs of different size, origin, and composition. Moreover, for the first time, the process of LD formation in the endothelium was detected and visualized in 3D. L ipid droplets (LDs) have been described in diverse cell types in histological and morphological studies for more than a 150 years. It is likely that structures referred to as “lipid bodies”, “lipid globules”, “lipid particles”, and “oil bodies” all represent lipid droplets. In most cell types, lipid droplets are usually less than 1 μm in size, although in hepatic steatosis, lipid droplets may reach 10 μm. 1 At present, lipid droplets are under intensive study due to the increasing recognition of their significant role in many aspects of health and disease. Their function in the field of cell biology, the prevalence in metabolic syndromes, obesity, steatosis, and atherosclerosis have prompted medical research on LDs. 2-6 Since many of these diseases manifest themselves by a considerable increase in lipid accumulation (e.g., in liver or arteries), leading to the formation of large lipid droplets far exceeding the size of individual cells, it is necessary to extend the study of lipid droplets to the tissue level. LDs can also be found in many cell types including hepatocytes and endothelial cells (ECs) as lipid-rich, spherical cytoplasmic inclusions with a distinctive architecture. In contrast to membranous organelles and cytoplasmic vesicles that have an aqueous content surrounded by a phospholipid bilayer membrane, LDs are composed of a neutral lipid core enclosed by a monolayer of phospholipids with associated proteins. 2,7 LDs were previously thought to be just an energy reservoir of the cell, but in recent years, it has become clear that they might have other functions. 2,3,8-10 LDs are common in organelles of eukaryotic cells, but their biogenesis is not fully under- stood. 11-13 It is accepted that LDs originate from the endoplasmic reticulum (ER), where neutral lipids are synthesized, and then, they aggregate within the hydrophobic core of the ER membrane. Upon reaching a critical size, LDs are thought to bud from the ER to form an independent Received: April 16, 2014 Accepted: June 3, 2014 Article pubs.acs.org/ac © XXXX American Chemical Society A dx.doi.org/10.1021/ac501395g | Anal. Chem. XXXX, XXX, XXX-XXX