Zooming into lipid droplet biology through the lens of electron microscopy.

Electron microscopy (EM), in its various flavors, has significantly contributed to our understanding of lipid droplets (LD) as central organelles in cellular metabolism. For example, EM has illuminated that LDs, in contrast to all other cellular organelles, are uniquely enclosed by a single phospholipid monolayer, revealed the architecture of LD contact sites with different organelles, and provided near-atomic resolution maps of key enzymes that regulate neutral lipid biosynthesis and LD biogenesis. In this review, we first provide a brief history of pivotal findings in LD biology unveiled through the lens of an electron microscope. We describe the main EM techniques used in the context of LD research and discuss their current capabilities and limitations, thereby providing a foundation for utilizing suitable EM methodology to address LD-related questions with sufficient level of structural preservation, detail, and resolution. Finally, we highlight examples where EM has recently been and is expected to be instrumental in expanding the frontiers of LD biology.

Lipophagy deficiency exacerbates ectopic lipid accumulation and tubular cells injury in diabetic nephropathy.

Autophagy-mediated lipotoxicity plays a critical role in the progression of diabetic nephropathy (DN), but the precise mechanism is not fully understood. Whether lipophagy, a selective type of autophagy participates in renal ectopic lipid deposition (ELD) and lipotoxicity in the kidney of DN is unknown. Here, decreased lipophagy, increased ELD and lipotoxcity were observed in tubular cells of patients with DN, which were accompanied with reduced expression of AdipoR1 and p-AMPK. Similar results were found in db/db mice, these changes were reversed by AdipoRon, an adiponectin receptor activator that promotes autophagy. Additionally, a significantly decreased level of lipophagy was observed in HK-2 cells, a human proximal tubular cell line treated with high glucose, which was consistent with increased lipid deposition, apoptosis and fibrosis, while were partially alleviated by AdipoRon. However, these effects were abolished by pretreatment with ULK1 inhibitor SBI-0206965, autophagy inhibitor chloroquine and enhanced by AMPK activator AICAR. These data suggested by the first time that autophagy-mediated lipophagy deficiency plays a critical role in the ELD and lipid-related renal injury of DN.

Comparative proteomics reveals that lipid droplet-anchored mitochondria are more sensitive to cold in brown adipocytes.

Brown adipose tissue (BAT) is specialized for uncoupled heat production through mitochondrion fueled majorly from fatty acids (FAs) of lipid droplets (LDs). How the interaction between the two organelles contributes the generation of heat remains elusive. Here, we report that LD-anchored mitochondria (LDAM) were observed in the BAT of mice raised at three different temperatures, 30 °C, 23 °C, and 6 °C. The biochemical analyses including Western blotting of electron transport chain subunits showed that LDAM were functional. Comparative proteomics analysis was conducted, which revealed differential expressions of proteins between LDAM and cytoplasmic mitochondria (CM) at different temperatures. Higher expressions of proteins at low temperature were observed for i) FA ß-oxidation in LDAM including FA synthesis and uncoupling, ii) pseudo-futile cycle in CM, and iii) two shuttle systems: glycerol 3-phosphate in both CM and LDAM and citrate malate in CM. Together, these results suggest that LDs and LDAM form a preorganized and functional organelle complex that permits the rapid response to cold.

Nuclear lipid droplets and nuclear damage in Caenorhabditis elegans.

Fat stored in the form of lipid droplets has long been considered a defining characteristic of cytoplasm. However, recent studies have shown that nuclear lipid droplets occur in multiple cells and tissues, including in human patients with fatty liver disease. The function(s) of stored fat in the nucleus has not been determined, and it is possible that nuclear fat is beneficial in some situations. Conversely, nuclear lipid droplets might instead be deleterious by disrupting nuclear organization or triggering aggregation of hydrophobic proteins. We show here that nuclear lipid droplets occur normally in C. elegans intestinal cells and germ cells, but appear to be associated with damage only in the intestine. Lipid droplets in intestinal nuclei can be associated with novel bundles of microfilaments (nuclear actin) and membrane tubules that might have roles in damage repair. To increase the normal, low frequency of nuclear lipid droplets in wild-type animals, we used a forward genetic screen to isolate mutants with abnormally large or abundant nuclear lipid droplets. Genetic analysis and cloning of three such mutants showed that the genes encode the lipid regulator SEIP-1/seipin, the inner nuclear membrane protein NEMP-1/Nemp1/TMEM194A, and a component of COPI vesicles called COPA-1/α-COP. We present several lines of evidence that the nuclear lipid droplet phenotype of copa-1 mutants results from a defect in retrieving mislocalized membrane proteins that normally reside in the endoplasmic reticulum. The seip-1 mutant causes most germ cells to have nuclear lipid droplets, the largest of which occupy more than a third of the nuclear volume. Nevertheless, the nuclear lipid droplets do not trigger apoptosis, and the germ cells differentiate into gametes that produce viable, healthy progeny. Thus, our results suggest that nuclear lipid droplets are detrimental to intestinal nuclei, but have no obvious deleterious effect on germ nuclei.

Single-shot quantitative phase imaging as an extension of differential interference contrast microscopy.

Various studies have been conducted to obtain quantitative phase information based on differential interference contrast (DIC) microscopy. As one such attempt, we propose in this study a single-shot quantitative phase imaging (QPI) method by combining two developments. First, an add-on optical system to a commercialized DIC microscope was developed to perform quantitative phase gradient imaging (QPGI) with single image acquisition using a polarization camera. Second, an algorithm was formulated to reconstitute QPI from the obtained QPGI by reducing linear artifacts, which arise in simply integrated QPGI images. To demonstrate the applicability of the developed system in cell biology, the system was used to measure various cell lines and compared with fluorescence microscopy images of the same field of view. Consistent with previous studies, nucleoli and lipid droplets can be imaged by the system with greater optical path lengths (OPL). The results also implied that combining fluorescence microscopy and the developed system might be more informative for cell biology research than using these methods individually. Exploiting the single-shot performance of the developed system, time-lapse imaging was also conducted to visualize the dynamics of intracellular granules in monocyte-/macrophage-like cells. Our proposed approach may accelerate the implementation of QPI in standard biomedical laboratories.

Three-dimensional ATUM-SEM reconstruction and analysis of hepatic endoplasmic reticulum‒organelle interactions.

The endoplasmic reticulum (ER) is a contiguous and complicated membrane network in eukaryotic cells, and membrane contact sites (MCSs) between the ER and other organelles perform vital cellular functions, including lipid homeostasis, metabolite exchange, calcium level regulation, and organelle division. Here, we establish a whole pipeline to reconstruct all ER, mitochondria, lipid droplets, lysosomes, peroxisomes, and nuclei by automated tape-collecting ultramicrotome scanning electron microscopy and deep learning techniques, which generates an unprecedented 3D model for mapping liver samples. Furthermore, the morphology of various organelles and the MCSs between the ER and other organelles are systematically analyzed. We found that the ER presents with predominantly flat cisternae and is knitted tightly all throughout the intracellular space and around other organelles. In addition, the ER has a smaller volume-to-membrane surface area ratio than other organelles, which suggests that the ER could be more suited for functions that require a large membrane surface area. Our data also indicate that ER‒mitochondria contacts are particularly abundant, especially for branched mitochondria. Our study provides 3D reconstructions of various organelles in liver samples together with important fundamental information for biochemical and functional studies in the liver.

Correlations of Fat Content in Human Milk with Fat Droplet Size and Phospholipid Species.

Fat globule size and phospholipid (PL) content in human milk (HM) were investigated. HM was classified into three groups depending on fat content (A < B < C). PL content (mg/100 g HM) was significantly higher in the C group (p < 0.05), indicating its positive relationship with HM fat content. When the PL content was normalized (mg/g fat), that of group A was significantly higher (p < 0.05) and fat droplet size in group C was slightly larger, suggesting that HM fat content is affected by fat droplet numbers to a larger extent than by fat droplet size. A correlation between PC and SM content in HM was observed regardless of fat content, while correlation between PE and either PC or SM increased in the order of C > B > A, hence the composition and content of PL species in HM varied according to its fat content.

Role of Constitutive STAR in Leydig Cells.

Leydig cells contain significant amounts of constitutively produced steroidogenic acute regulatory protein (STAR; STARD1). Hormone-induced STAR plays an essential role in inducing the transfer of cholesterol into the mitochondria for hormone-dependent steroidogenesis. STAR acts at the outer mitochondrial membrane, where it interacts with a protein complex, which includes the translocator protein (TSPO). Mutations in STAR cause lipoid congenital adrenal hyperplasia (lipoid CAH), a disorder characterized by severe defects in adrenal and gonadal steroid production; in Leydig cells, the defects are seen mainly after the onset of hormone-dependent androgen formation. The function of constitutive STAR in Leydig cells is unknown. We generated STAR knockout (KO) MA-10 mouse tumor Leydig cells and showed that STAR KO cells failed to form progesterone in response to dibutyryl-cAMP and to TSPO drug ligands, but not to 22(R)-hydroxycholesterol, which is a membrane-permeable intermediate of the CYP11A1 reaction. Electron microscopy of STAR KO cells revealed that the number and size of lipid droplets were similar to those in wild-type (WT) MA-10 cells. However, the density of lipid droplets in STAR KO cells was drastically different than that seen in WT cells. We isolated the lipid droplets and analyzed their content by liquid chromatography-mass spectrometry. There was a significant increase in cholesteryl ester and phosphatidylcholine content in STAR KO cell lipid droplets, but the most abundant increase was in the amount of diacylglycerol (DAG); DAG 38:1 was the predominantly affected species. Lastly, we identified genes involved in DAG signaling and lipid metabolism which were differentially expressed between WT MA-10 and STAR KO cells. These results suggest that constitutive STAR in Leydig cells is involved in DAG accumulation in lipid droplets, in addition to cholesterol transport. The former event may affect cell functions mediated by DAG signaling.

Evidences for lipid involvement in SARS-CoV-2 cytopathogenesis.

The pathogenesis of SARS-CoV-2 remains to be completely understood, and detailed SARS-CoV-2 cellular cytopathic effects requires definition. We performed a comparative ultrastructural study of SARS-CoV-1 and SARS-CoV-2 infection in Vero E6 cells and in lungs from deceased COVID-19 patients. SARS-CoV-2 induces rapid death associated with profound ultrastructural changes in Vero cells. Type II pneumocytes in lung tissue showed prominent altered features with numerous vacuoles and swollen mitochondria with presence of abundant lipid droplets. The accumulation of lipids was the most striking finding we observed in SARS-CoV-2 infected cells, both in vitro and in the lungs of patients, suggesting that lipids can be involved in SARS-CoV-2 pathogenesis. Considering that in most cases, COVID-19 patients show alteration of blood cholesterol and lipoprotein homeostasis, our findings highlight a peculiar important topic that can suggest new approaches for pharmacological treatment to contrast the pathogenicity of SARS-CoV-2.

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.

Dietary Carbohydrates and Fat Induce Distinct Surfactant Alterations in Mice.

Obesity and type 2 diabetes are nutrition-related conditions associated with lung function impairment and pulmonary diseases; however, the underlying pathomechanisms are incompletely understood. Pulmonary surfactant is essential for lung function, and surfactant synthesis by AT2 (alveolar epithelial type 2) cells relies on nutrient uptake. We hypothesized that dietary amounts of carbohydrates or fat affect surfactant homeostasis and composition. Feeding mice a starch-rich diet (StD), sucrose-rich diet (SuD), or fat-rich diet (FaD) for 30 weeks resulted in hypercholesterolemia and hyperinsulinemia compared with a fiber-rich control diet. In SuD and FaD groups, lung mechanic measurements revealed viscoelastic changes during inspiration, indicating surfactant alterations, and interfacial adsorption of isolated surfactant at the air-liquid interface was decreased under FaD. The composition of characteristic phospholipid species was modified, including a shift from dipalmitoyl-phosphatidylcholine (PC16:0/16:0) to palmitoyl-palmitoleoyl-phosphatidylcholine (PC16:0/16:1) in response to carbohydrates and decreased myristic acid-containing phosphatidylcholine species (PC14:0/14:0; PC16:0/14:0) on excess fat intake, as well as higher palmitoyl-oleoyl-phosphatidylglycerol (PG16:0/18:1) and palmitoyl-linoleoyl-phosphatidylglycerol (PG16:0/18:2) fractions in StD, SuD, and FaD groups than in the control diet. Moreover, mRNA expression levels of surfactant synthesis-related proteins within AT2 cells were altered. Under the StD regimen, AT2 cells showed prominent lipid accumulations and smaller lamellar bodies. Thus, in an established mouse model, distinct diet-related surfactant alterations were subtle, yet detectable, and may become challenging under conditions of reduced respiratory capacity. Dietary fat was the only macronutrient significantly affecting surfactant function. This warrants future studies examining alimentary effects on lung surfactant, with special regard to pulmonary complications in obesity and type 2 diabetes.

Nuclear lipid droplets form in the inner nuclear membrane in a seipin-independent manner.

Nuclear lipid droplets (LDs) in hepatocytes are derived from precursors of very-low-density lipoprotein in the ER lumen, but it is not known how cells lacking the lipoprotein secretory function form nuclear LDs. Here, we show that the inner nuclear membrane (INM) of U2OS cells harbors triglyceride synthesis enzymes, including ACSL3, AGPAT2, GPAT3/GPAT4, and DGAT1/DGAT2, and generates nuclear LDs in situ. mTOR inhibition increases nuclear LDs by inducing the nuclear translocation of lipin-1 phosphatidic acid (PA) phosphatase. Seipin, a protein essential for normal cytoplasmic LD formation in the ER, is absent in the INM. Knockdown of seipin increases nuclear LDs and PA in the nucleus, whereas seipin overexpression decreases these. Seipin knockdown also up-regulates lipin-1ß expression, and lipin-1 knockdown decreases the effect of seipin knockdown on nuclear LDs without affecting PA redistribution. These results indicate that seipin is not directly involved in nuclear LD formation but instead restrains it by affecting lipin-1 expression and intracellular PA distribution.

Rational construction of AIEgens with wide color tunability and their specific lipid droplet imaging applications.

Organic light-emitting materials with aggregation-induced emission (AIE) character have experienced a rapid development in imaging, visualization and sensing. In this paper, by installing rotors to an aggregation-caused quenching (ACQ) compound, we constructed an AIE luminogen (AIEgens) system (A2-A4, B1-B3) with wide emission tunability, which covers almost all regions of visible light (400-780 nm). The calculated energy gap of the compounds is consistent with the value of the absorption transition. In the biological experiments, B3 showed excellent lipid droplet (LD) targeting ability in PC12 cells, and higher photostability compared with the commercial LDs bioprobe. Moreover, as a hydrophobic compound, B3 can distinguish blood from a hyperlipidemia patient and normal people. Thus, this study provides a strategy to construct red/NIR fluorescent materials with AIE character, and further apply it to identify the blood of people with hyperlipidemia.

Fatty acid binding protein 7 mediates linoleic acid-induced cell death in triple negative breast cancer cells by modulating 13-HODE.

Different fatty acids have distinct effects on the survival of breast cancer cells, which could be mediated by fatty acid binding proteins (FABPs), a family of lipid chaperones. Due to the diverse structures of the members of FABP family, each FABP demonstrates distinct binding affinities to different fatty acids. Of note, FABP7 is predominantly expressed in triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer. Yet, the role of FABP7 in modulating the effects of fatty acids on TNBC survival was unclear. In contrast to the high expression of FABP7 in human TNBC tumours, FABP7 protein was undetectable in TNBC cell lines. Hence, a FABP7 overexpression model was used for this study, in which the transduced TNBC cell lines (MDA-MB-231 and Hs578T) were treated with various mono- and polyunsaturated fatty acids. Oleic acid (OA), docosahexaenoic acid (DHA) and arachidonic acid (AA) inhibited TNBC cell growth at high concentrations, with no differences resulted from FABP7 overexpression. Interestingly, overexpression of FABP7 augmented linoleic acid-induced cell death in MDA-MB-231 cells. The increased cell death may be explained by a decrease in 13-HODE, a pro-tumorigenic oxidation product of linoleic acid. The phenotype was, however, attenuated with a rescue treatment using 25 nM 13-HODE. The decrease in 13-HODE was potentially due to fatty acid partitioning modulated by FABP7, as demonstrated by a 3-fold increase in fatty acid oxidation. Our findings suggest that linoleic acid could be a potential therapeutic strategy for FABP7-overexpressing TNBC patients.

Loss of the seipin gene perturbs eggshell formation in Caenorhabditiselegans.

Seipin, an evolutionary conserved protein, plays pivotal roles during lipid droplet (LD) biogenesis and is associated with various human diseases with unclear mechanisms. Here, we analyzed Caenorhabditis elegans mutants deleted of the sole SEIPIN gene, seip-1 Homozygous seip-1 mutants displayed penetrant embryonic lethality, which is caused by the disruption of the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell. In C. elegans oocytes and embryos, SEIP-1 is associated with LDs and is crucial for controlling LD size and lipid homeostasis. The seip-1 deletion mutants reduced the ratio of polyunsaturated fatty acids (PUFAs) in their embryonic fatty acid pool. Interestingly, dietary supplementation of selected n-6 PUFAs rescued the embryonic lethality and defective permeability barrier. Accordingly, we propose that SEIP-1 may maternally regulate LD biogenesis and lipid homeostasis to orchestrate the formation of the permeability barrier for eggshell synthesis during embryogenesis. A lipodystrophy allele of seip-1 resulted in embryonic lethality as well and could be rescued by PUFA supplementation. These experiments support a great potential for using C. elegans to model SEIPIN-associated human diseases.

A Framework for Optimizing High-Content Imaging of 3D Models for Drug Discovery.

Three-dimensional (3D) spheroid models are rapidly gaining favor for drug discovery applications due to their improved morphological characteristics, cellular complexity, long lifespan in culture, and higher physiological relevance relative to two-dimensional (2D) cell culture models. High-content imaging (HCI) of 3D spheroid models has the potential to provide valuable information to help researchers untangle disease pathophysiology and assess novel therapies more effectively. The transition from 2D monolayer models to dense 3D spheroids in HCI applications is not trivial, however, and requires 3D-optimized protocols, instrumentation, and resources. Here, we discuss considerations for moving from 2D to 3D models and present a framework for HCI and analysis of 3D spheroid models in a drug discovery setting. We combined scaffold-free, multicellular spheroid models with scalable, automation-compatible plate technology enabling image-based applications ranging from high-throughput screening to more complex, lower-throughput microphysiological systems of organ networks. We used this framework in three case studies: investigation of lipid droplet accumulation in a human liver nonalcoholic steatohepatitis (NASH) model, real-time immune cell interactions in a multicellular 3D lung cancer model, and a high-throughput screening application using a 3D co-culture model of gastric carcinoma to assess dose-dependent drug efficacy and specificity. The results of these proof-of-concept studies demonstrate the potential for high-resolution image-based analysis of 3D spheroid models for drug discovery applications, and confirm that cell-level and temporal-spatial analyses that fully exploit multicellular features of spheroid models are not only possible but soon will be routine practice in drug discovery workflows.

The biology of lipid droplet-bound mitochondria.

Proper regulation of cellular lipid storage and oxidation is indispensable for the maintenance of cellular energy homeostasis and health. Mitochondrial function has been shown to be a main determinant of functional lipid storage and oxidation, which is of particular interest for the adipose tissue, as it is the main site of triacylglyceride storage in lipid droplets (LDs). Recent studies have identified a subpopulation of mitochondria attached to LDs, peridroplet mitochondria (PDM) that can be separated from cytoplasmic mitochondria (CM) by centrifugation. PDM have distinct bioenergetics, proteome, cristae organization and dynamics that support LD build-up, however their role in adipose tissue biology remains largely unexplored. Therefore, understanding the molecular basis of LD homeostasis and their relationship to mitochondrial function and attachment in adipocytes is of major importance.

Magnesium isoglycyrrhizinate prevents the nonalcoholic hepatic steatosis via regulating energy homeostasis.

Non-alcoholic fatty liver disease is a public health problem worldwide associated with high morbidity and hepatic steatosis, but no effective therapeutic interventions. Magnesium isoglycyrrhizinate (MGIG), a derivative of an active component of Glycyrrhiza glabra, is widely used for the treatment of inflammatory liver diseases due to its potent anti-inflammatory and hepatoprotective activities. Hence, this study aimed to study the effects of MGIG on hepatic steatosis in mice fed a high-fat diet (HFD). Oil Red O staining and transmission electron microscopy revealed a decrease in lipid accumulation in the liver after MGIG treatment along with improved mitochondrial ultramicrostructures. Metabonomic analysis demonstrated that MGIG intervention increased glutamate utilization in mitochondria by promoting the uptake of glutamate into the tricarboxylic acid (TCA) cycle. The NAD+ /NADH ratio and the expression of other lipid-metabolism-related genes were increased in MGIG-treated livers. Transcriptome sequencing showed that the expression of TLR4, an isoform of the innate immunity Toll-like receptors (TLRs), was significantly decreased after MGIG treatment, suggesting a link between the anti-inflammatory effects of MGIG and its suppression of lipidation. Our results reveal the potent effects of MGIG on lipid metabolism and suggest that hepatic TLR4 might be a crucial therapeutic target to regulate energy homeostasis in hepatic steatosis.

Relative lipid oxidation associates directly with mitochondrial fusion phenotype and mitochondria-sarcoplasmic reticulum interactions in human skeletal muscle.

Disturbances in skeletal muscle lipid oxidation might induce ectopic fat deposition and lipotoxicity. Nevertheless, the cellular mechanisms that regulate skeletal muscle lipid oxidation have not been fully determined. We aimed to determine whether there was an association between relative whole body lipid oxidation and mitochondrial size or mitochondria-sarcoplasmic reticulum interactions in the skeletal muscle. Twelve healthy men were included [mean (standard deviation), 24.7 (1.5) yr old, 24.4 (2.6) kg/m2]. The respiratory quotient (RQ) was used to estimate relative lipid oxidation at rest and during exercise (50% maximal oxygen consumption, 600 kcal expended). A skeletal muscle biopsy was obtained from the vastus lateralis at rest. Transmission electron microscopy was used to determine mitochondrial size and mitochondria-sarcoplasmic reticulum interactions (≤50 nm of distance between organelles). Protein levels of fusion/fission regulators were measured in skeletal muscle by Western blot. Resting RQ and exercise RQ associated inversely with intermyofibrillar mitochondrial size (r = -0.66 and r = -0.60, respectively, P < 0.05). Resting RQ also associated inversely with the percentage of intermyofibrillar mitochondria-sarcoplasmic reticulum interactions (r = -0.62, P = 0.03). Finally, intermyofibrillar mitochondrial size associated inversely with lipid droplet density (r = -0.66, P = 0.01) but directly with mitochondria fusion-to-fission ratio (r = 0.61, P = 0.03). Our results show that whole body lipid oxidation is associated with skeletal muscle intermyofibrillar mitochondrial size, fusion phenotype, and mitochondria-sarcoplasmic-reticulum interactions in nondiabetic humans.

Synchrotron multimodal imaging in a whole cell reveals lipid droplet core organization.

A lipid droplet (LD) core of a cell consists mainly of neutral lipids, triacylglycerols and/or steryl esters (SEs). The structuration of these lipids inside the core is still under debate. Lipid segregation inside LDs has been observed but is sometimes suggested to be an artefact of LD isolation and chemical fixation. LD imaging in their native state and in unaltered cellular environments appears essential to overcome these possible technical pitfalls. Here, imaging techniques for ultrastructural study of native LDs in cellulo are provided and it is shown that LDs are organized structures. Cryo soft X-ray tomography and deep-ultraviolet (DUV) transmittance imaging are showing a partitioning of SEs at the periphery of the LD core. Furthermore, DUV transmittance and tryptophan/tyrosine auto-fluorescence imaging on living cells are combined to obtain complementary information on cell chemical contents. This multimodal approach paves the way for a new label-free organelle imaging technique in living cells.