This research was funded by National and European Funds via the Portuguese Science and Technology Foundation (FCT), European Regional Development Fund (FEDER), and Programa Operacional Factores de Competitividade (COMPETE): 2020.09481.BD, UIDP/04539/2020 (CIBB), and POCI-01-0145-FEDER-007440. The authors would like to thank the support of iLAB - Microscopy and Bioimaging Lab, a facility of the Faculty of Medicine of the University of Coimbra and a member of the national infrastructure PPBI-Portuguese Platform of BioImaging (POCI-01-0145-FEDER-022122), as well as support from FSE CENTRO-04-3559-FSE-000142.

All animal procedures performed in this study were approved by the Coimbra Institute for Clinical and Biomedical Research (iCBR) Animal Welfare Body (ORBEA, #9/2018) and complied with the Animal Care National and European Directives and with the ARRIVE guidelines.
1. Experimental design
<ol>
	<li>Pair-house 13 week old male Wistar rats in ventilated cages under controlled environmental conditions of temperature (22 &#176;C &#177; 1 &#176;C), humidity (50%-60%), and light (12 h light-dark cycle) and with ad libitum access to tap water and standard rodent chow.</li>
	<li>After a 2 week period of acclimatization, arbitrarily assign the rats into two groups.</li>
	<li>Feed the control (CTRL, n = 6) and high-fat diet groups (HFD, n = 6) with standard chow and a high-fat diet (45% kcal/fat), respectively, for 24 weeks.</li>
	<li>Monitor the body weight (BW) weekly. Record the food and beverage consumption daily per cage.</li>
</ol>
2. Liver sample preparation
<ol>
	<li>Liver dissection
	<ol>
		<li>Prepare the peristaltic pump: Run 70% ethanol through the tubing, connect a 27 G needle to the outlet end of the tubing, and prime the tubing with ice-cold (4 &#176;C) 1x phosphate-buffered saline (PBS, pH ~7.4 ) (e.g., 200 rpm on a peristaltic pump with 1.6 mm I.D. silicon tubing, IV mini drip set). Adjust for the body weight (e.g., for a 100-150 g rat, use a flow rate of approximately 10-12 mL/min).</li>
		<li>Anesthetize the rat by an intraperitoneal injection using an anesthesia protocol (the final concentration of ketamine = 75 mg/kg, and the final concentration of medetomidine = 1 mg/kg). 
		NOTE: Make sure the rat is completely anesthetized using the toe pinch response method.</li>
		<li>Place the rat on the dissection tray.</li>
		<li>Sanitize the fur thoroughly with 70% ethanol, and dry it with a paper towel.</li>
		<li>Using scissors, make a &#34;U&#34;-shaped incision through the skin, and cut open below the diaphragm. Then, cut the rib cage rostrally on the edges to expose the heart.</li>
		<li>While the 1x PBS is running, pass the needle through the left ventricle into the ascending aorta, and clamp. Then, the right atrium is cut to allow drainage once the perfusion has begun. 
		NOTE: The liver should begin to blanch as blood is replaced with PBS.</li>
		<li>Using scissors and Dumont forceps, remove the liver carefully, and rinse with 1x PBS.</li>
		<li>Transfer the liver onto a Petri dish, and weigh it.</li>
		<li>Using a scalpel, collect hepatic tissue samples of 5 mm in thickness from the lateral side (1 cm from the edge) of the left lobe of the liver for the embedding process. 
		NOTE The remaining tissue can be stored at &#8722;80 &#176;C for long-term storage.</li>
	</ol>
	</li>
	<li>Liver tissue embedding
	<ol>
		<li>Prepare a dry ice container, and properly label cryomolds with the sample ID and orientation.</li>
		<li>Place a few drops of cryo embedding matrix onto the center of the cryomold to make the optimal cutting temperature (OCT).</li>
		<li>Ensure the tissue sample is properly oriented for a transverse section. 
		NOTE: Make sure that the side touching the bottom of the cryomold is the side that will be sectioned first.</li>
		<li>Carefully drop more OCT onto the cryomold until it is completely covered. Try to avoid the formation of air bubbles. If necessary, with plain forceps, remove any bubbles inside the OCT.</li>
		<li>Rapidly place the cryomold with the OCT-covered sample in a dry ice container. 
		&#8203;NOTE: The tissues can be stored at &#8722;80 &#176;C for 3 years.</li>
	</ol>
	</li>
</ol>
3. Frozen tissue sectioning
<ol>
	<li>Adjust the cryostat temperature (chamber: &#8722;21 &#176;C; specimen: &#8722;18 &#176;C), and insert a new sterile blade.</li>
	<li>Place the samples in the cryostat for 30 min before allowing the temperature to equilibrate.</li>
	<li>Make a single layer of OCT on the specimen disc to allow sample adhesion. Allow the OCT to freeze, and mount the tissue sample in the desired orientation. 
	NOTE: The cutting surface should be parallel to the blade. To maintain good adhesion, place the heat extractor on the top of the sample. The previous steps can also be performed in a dry ice container with dry ice.</li>
	<li>Place the sample in the specimen head, and trim the surface of the tissue (a few 30 &#181;m tissue sample sections).</li>
	<li>Once the region of interest is accessible for sectioning, cut 12 &#181;m thick sections, and place them onto labeled microscope slides kept at room temperature (RT). 
	NOTE: To collect the section, slowly move the slide toward the tissue section. Each slide can collect two liver sections.</li>
	<li>Allow the slides to dry for 10 min at RT. 
	&#8203;NOTE: The slides can be stored at &#8722;20 &#176;C for 6-12 months or at &#8722;80 &#176;C for up to 3 years.</li>
</ol>
4. BODIPY staining
<ol>
	<li>Thaw the slides in a slide staining system at RT for 30 min.</li>
	<li>Wash with 1x PBS (3x for 5 min each).</li>
	<li>Circle the section with a hydrophobic layer using a barrier pen.</li>
	<li>Prepare 500 &#181;g/mL BODIPY 493/503&#160;stock solution: Dissolve 1 mg of BODIPY 493/503in 2 mL of solvent (90% DMSO; 10% 1x PBS). Protect from light. Sonicate in an ultrasonic bath for 1 h (9.5-10 W) at 37 &#176;C. 
	NOTE: The solution can be stored at &#8722;20 &#176;C for at least 30 days.</li>
	<li>Prepare the BODIPY staining solution (1 &#181;g/mL) by adding 2 &#181;L of BODIPY 493/503 stock solution and 0.1 &#181;L of DAPI stock solution (5 mg/mL) to 997.9 &#181;L of 1x PBS.</li>
	<li>Incubate the slides (75 &#181;L/slide) with BODIPY 493/503 (1 &#181;g/mL) and DAPI (0.1 &#181;g/mL) at RT for 40 min. 
	NOTE: Keep the slides in the dark from this step onward.</li>
	<li>Wash the slides with 1x PBS (3x for 5 min each).</li>
	<li>Mount the slides with coverslips using a fluorescence mounting medium, allow them to dry for 30 min, and seal them with nail polish. 
	&#8203;NOTE: The slides can be stored at 4 &#176;C until imaging.</li>
</ol>
5. Lipid quantification
<ol>
	<li>Measure the serum total cholesterol and TG levels using an automatic, validated method and equipment.</li>
	<li>Measure the hepatic TG levels using a triglycerides colorimetric assay kit (Table of Materials) according to the manufacturer&#39;s protocol.</li>
</ol>
6. Image acquisition
<ol>
	<li>Place the slide on the laser scanning confocal microscope slide holder.</li>
	<li>For image visualization and acquisition, use a confocal microscope with a 20x objective lens (plan-apochromat: 20x/0.8).</li>
	<li>To avoid cross-talk between the BODIPY 493/503 and DAPI, use the sequential (best signal) scan mode on the confocal software.</li>
	<li>Excite the BODIPY 593/503 using the 488 nm argon laser line and the DAPI using the 405 nm laser line. Set the emission ranges at 493-589 nm for BODIPY 493/503 and at 410-464 nm for DAPI.</li>
	<li>Use the following settings: pinhole: 1 AU, resolution: 1,024 pixels x 1,024 pixels, bit depth: 12, pixel size: 0.415 &#181;m, bidirectional mode, scan speed: 7 (~1.58 &#181;s/pixel for a 20x objective), line averaging: 2x, and digital zoom: 1. 
	NOTE: The abovementioned scanning parameters must be optimized for each confocal microscope and objective used.</li>
	<li>Set the gain and digital gain appropriately so that no saturated pixels are detected on the range indicator. 
	NOTE: Correct the background signal by adjusting the offset.</li>
	<li>Once the LDs are correctly identified, acquire the image with the BODIPY and DAPI channels. 
	NOTE: All the pictures must be acquired in the same conditions (exposure and general settings) for each color channel.</li>
	<li>To create wide-area images (Figure 2), change the objective to a10x objective lens (plan-neofluar: 10x/0.3).</li>
	<li>Select Tile Scan mode, and make mosaics of 5 per 5 images. 
	NOTE: In this work, each tile had an overlap of 10% in order to appropriately merge the tiles for analysis.</li>
	<li>To create 3D and orthogonal views (Figure 3A), place a drop of immersion oil on the top of the cover glass, and change the objective to a 40x objective lens (plan-neofluar: 40x/1.30 oil).</li>
	<li>Select Z-Stack mode, and by adjusting the Z plane, define the first and last positions for acquisition with an optical slice at an optimal thickness (~0.5 &#181;m) to ensure that all the droplets are captured. 
	NOTE: To speed up the image acquisition and avoid bleaching, the optical slice can be adjusted to a suboptimal size, ensuring at least 30% oversampling for a good 3D reconstruction.</li>
	<li>Select the Ortho module, and create orthogonal views.</li>
	<li>Acquire 3D images by selecting the 3D module following Transparency Rendering Mode with the confocal microscope software.</li>
</ol>
7. Analysis of images
<ol>
	<li>Process and analyze the single-plane images (20x magnification) with CellProfiler (version 4.2.5). 
	NOTE: The pipeline used in this work was adapted from Adomshick et al.45.</li>
	<li>Click on the Images module in the top-left corner of the CellProfiler window, and upload the images as .tiff files.</li>
	<li>Use the NamesAndTypes module to sort between BODIPY- (droplet) and DAPI- (nuclei) stained images based on the file names. Perform the LD analysis only with BODIPY-stained images.</li>
	<li>To start the pipeline constrution, click on Adjust Modules, and select the module ColorToGray to convert the images to grayscale images. 
	NOTE: To identify objects, grayscale images are required.</li>
	<li>Identify droplets by employing the IdentifyPrimaryObjects module using the grayscale image. 
	NOTE: The parameters in this module must be adjusted to accomplish good LD identification. In this work, defining LDs with a size between 6 pixels and 300 pixels and a threshold correction factor of 1.0 led to the most accurate identification.</li>
	<li>To measure the pixel intensity of the identified lipid droplets, add a MeasureObjectIntensity module.</li>
	<li>Add an additional FilterObjects module to ensure that only the strongest signals are quantified while less intense signals are excluded from the final lipid droplet analysis (minimum intensity: 0.15; maximum intensity:1 arbitrary unit).</li>
	<li>To measure the LDs related to the output data, add the MeasureObjectSizeShape module.</li>
	<li>Add an OverlayOutlines module at this point to overlay the identified droplet on the original image and, thus, ensure that the segmentation looks accurate on the unprocessed image as well. 
	NOTE: This is an optional (quality control) step.</li>
	<li>Add an ExportToSpreadsheet module at the end, and click on the Analyze Images button at the bottom-left corner.</li>
</ol>
8. Statistical analysis
<ol>
	<li>Express the results as mean &#177; standard error of the mean (S.E.M.) using any statistical analysis software application. 
	NOTE: In this study, GraphPad software was used for statistical analysis.</li>
	<li>Analyze the distribution of values using the Kolmogorov-Smirnov test to assess significant deviations from normality. Analyze the parametric data using the Student&#39;s unpaired t-test. 
	NOTE: Values of p &#60; 0.05 were considered statistically significant.</li>
</ol>

Assessing Hepatic Steatosis with 3D Fluorescence Lipid Droplet Reconstruction

The authors have nothing to disclose.

This BODIPY 493/503 fluorescence-based protocol for LD assessment aimed to develop a new imaging approach for the evaluation of hepatic steatosis. Given the strong correlation between obesity and fatty liver disease, the Western-style high-fat diet was used to establish an animal model of hepatic steatosis26. A robust increase in hepatic TG contents was confirmed by a quantitative triglycerides colorimetric assay kit, which suggested a heightened hepatic lipidosis scenario in the HFD-fed animals. ...

Lipid droplets (LDs), classically viewed as energy depots, are specialized cellular organelles that mediate lipid storage, and they comprise a hydrophobic neutral lipid core, which mainly contains cholesterol esters and triglycerides (TGs), encapsulated by a phospholipid monolayer1,2,3.
LD biogenesis occurs in the endoplasmic reticulum (ER), starting with the synthesis of triacylglycerol (TAG) and sterol esters. Neutral lipids are diffused between the leaflets of the ER bilayer at low concentrations but coalesce into oil lenses that grow and bud into nearly spherical droplets from ER membrane when their intracellular concentration increases4. Subsequently, proteins from the ER bilayer and cytosol, particularly the perilipin (PLIN) protein family, translocate to the surfaces of the LDs to facilitate budding5,6,7,8,9.
Through new fatty acid synthesis and LD fusion or coalescence, LDs grow into different sizes. Accordingly, the size and number of LDs differ considerably across different cell types. Small droplets (300-800 nm diameter), which are known as initial LDs (iLDs), can be formed by nearly all cells4. Later in LD formation, most cells are able to convert some iLDs into larger ones-expanding LDs (eLDs &#62;1 &#181;m in diameter). Yet, just specific cell types, such as adipocytes and hepatocytes, have the capacity to form giant or supersized LDs (up to tens of microns in diameter)4,10.
LDs play a very important role in the regulation of cellular lipid metabolism, suppressing lipotoxicity, and preventing ER stress, mitochondrial dysfunction, and, ultimately, cell death caused by free fatty acids (FAs)11,12,13,14. Furthermore, LDs have also been implicated in the regulation of gene expression, viral replication protein sequestration, and membrane trafficking and signaling15,16,17. Therefore, the misregulation of LD biogenesis is a hallmark of chronic diseases associated with metabolic syndrome, obesity, type 2 diabetes mellitus (T2DM), and/or arteriosclerosis, to name just a few18,19,20.
The liver, as a metabolic hub, is mostly responsible for lipid metabolism by storing and processing lipids, and, therefore, it is constantly threatened by lipotoxicity21. Hepatic steatosis (HS) is a common feature of a series of progressive liver diseases and is characterized by excessive intracellular lipid accumulation in the form of cytosolic LDs that, ultimately, may lead to liver metabolic dysfunction, inflammation, and advanced forms of nonalcoholic fatty liver disease22,23,24,25. HS occurs when the rate of fatty acid oxidation and export as triglycerides within very low-density lipoproteins (VLDLs) is lower than the rate of hepatic fatty acid uptake from the plasma and de novo fatty acid synthesis26. The hepatic accumulation of lipids often occurs in two forms-microvesicular and macrovesicular steatosis-and these display distinct cytoarchitectonic characteristics27. Typically, microvesicular steatosis is characterized by the presence of small LDs dispersed throughout the hepatocyte with the nucleus placed centrally, whereas macrovesicular steatosis is characterized by the presence of a single large LD that occupies the greater part of the hepatocyte, pushing the nucleus to the periphery28,29. Notably, these two types of steatosis are often found together, and it remains unclear how these two LD patterns influence disease pathogenesis, as evidence is still inconsistent31,32,33,34. Yet, such type of analysis is often employed as a &#34;reference standard&#34; in preclinical and clinical studies to understand the dynamic behavior of LDs and characterize hepatic steatosis29,34,35,36.
Liver biopsies, the gold standard for diagnosing and grading HS, are routinely assessed by histological hematoxylin and eosin (H&#38;E) analysis, where lipid droplets are evaluated as unstained vacuoles in H&#38;E-stained liver sections37. While acceptable for macrovesicular steatosis evaluation, this type of staining generally narrows the assessment of microvesicular steatosis38. Lipid-soluble diazo dyes, such as Oil Red O (ORO), are classically combined with brightfield microscopy to analyze intracellular lipid stores, but these still have a number of disadvantages: (i) the usage of ethanol or isopropanol in the staining process, which often causes the disruption of the native LDs and occasional fusion despite the cells being fixed39; (ii) the time-consuming nature, as ORO solution requires fresh powder dissolving and filtering due to the limited shelf life, thus contributing to less consistent results; (iii) and the fact that ORO stains more than just lipid droplets and often overestimates hepatic steatosis38.
Consequently, cell-permeable lipophilic fluorophores, such as Nile Red, have been used in either live or fixed samples to overcome some of the aforementioned limitations. However, the non-specific nature of cellular lipid organelle labeling repeatedly narrows LD assessments40. Moreover, the spectral properties of Nile Red vary according to the polarity of the environment, which can often lead to spectral shifts41.
The lipophilic fluorescent probe 1,3,5,7,8-pentamethyl-4-bora-3a,4adiaza-s-indacene (excitation wavelength: 480 nm; emission maximum: 515 nm; BODIPY 493/503) exhibits hydrophobicity characteristics that allow its rapid uptake by intracellular LDs, accumulates in the lipid droplet core, and, subsequently, emits bright green fluorescence12. Unlike Nile Red, BODIPY 493/503 is insensitive to the environment polarity and has been shown to be more selective, as it displays high brightness for LD imaging. In order to stain neutral LDs, this dye can be used in live or fixed cells and successfully coupled with other staining and/or labeling methods42. Another advantage of the dye is that it requires little effort to place into a solution and is stable, thus eliminating the need to freshly prepare it for each experiment42. Even though the BODIPY 493/503 probe has been successfully employed to visualize the localization and dynamics of LDs in cell cultures, some reports have also demonstrated the reliable use of this dye as an LD imaging tool in tissues including the human vastus lateralis muscle43, the rat soleus muscle42, and the mouse intestine44.
Herein, we propose an optimized BODIPY 493/503-based protocol as an alternative analytical approach for the evaluation of the LD number, area, and diameter in liver specimens from an animal model of hepatic steatosis. This procedure covers liver sample preparation, tissue sectioning, staining conditions, image acquisition, and data analysis.

<table><tbody><tr><td>1.6 mm I.D. silicone tubing, I.V mini drip set</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>4,4-difluoro-1,3,5,7,8-pentametil-4-bora-3a,4a-diaza-s-indaceno (BODIPY 493/503)</td><td>Sigma-Aldrich, Lyon, France</td><td>D3922</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Molecular Probes Inc, Invitrogen, Eugene, OR</td><td>D1306</td><td></td></tr><tr><td>70% ethanol</td><td>Honeywell</td><td>10191455</td><td></td></tr><tr><td>Adobe Illustrator CC</td><td>Adobe Inc.</td><td>Used to design the figures</td><td></td></tr><tr><td>Automatic analyzer Hitachi 717</td><td>Roche Diagnostics Inc., Mannheim, Germany</td><td>8177-30-0010</td><td></td></tr><tr><td>Barrier pen (Liquid blocker super pap pen)</td><td>Daido Sangyo Co., Ltd, Japon</td><td>_</td><td></td></tr><tr><td>Blade</td><td>Leica</td><td>221052145</td><td>Used in the cryostat</td></tr><tr><td>Cell Profiler version 4.2.5</td><td>https://cellprofiler.org/releases/</td><td>Used to analyse the acquired images</td><td></td></tr><tr><td>Coverslips</td><td>Menzel-Glaser, Germany</td><td>_</td><td></td></tr><tr><td>Cryomolds</td><td>Tissue-Tek</td><td>_</td><td></td></tr><tr><td>Cryostat (including specimen disc and heat extractor) CM3050 S</td><td>Leica Biosystems</td><td>_</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich, Lyon, France</td><td>D-8418</td><td>Used to dissolve Bodipy for the 5 mg/mL stock solution. CAUTION: Toxic&lt;br/&gt; and flammable. Vapors may cause&lt;br/&gt; irritation. Manipulate in a fume&lt;br/&gt; hood. Avoid direct contact with skin.&lt;br/&gt; Wear rubber gloves, protective eye&lt;br/&gt; goggles.</td></tr><tr><td>Dry ice container (styrofoam cooler)</td><td>Novolab</td><td>A26742</td><td></td></tr><tr><td>Dumont forceps</td><td>Fine Science Tools, Germany</td><td>11295-10</td><td></td></tr><tr><td>Glass Petri dish (H 25 mm, &amp;oslash;&lt;br/&gt; 150 mm)</td><td>Thermo Scientific</td><td>150318</td><td>Used to weigh the liver after dissection</td></tr><tr><td>Glycergel</td><td>DAKO Omnis</td><td>S303023</td><td></td></tr><tr><td>GraphPad Prism software, version 9.3.1</td><td>GraphPad Software, Inc., La Jolla, CA, USA</td><td></td><td></td></tr><tr><td>High-fat diet</td><td>Envigo, Barcelona, Spain</td><td>MD.08811</td><td></td></tr><tr><td>Ketamine (Nimatek&amp;nbsp; 100 mg/mL)</td><td>Dechra</td><td>791/01/14DFVPT</td><td>Used at a final concentration of 75 mg/kg</td></tr><tr><td>Laser scanning confocal microscope&amp;nbsp; (QUASAR detection unit; )</td><td>Carl Zeiss, germany</td><td>LSM 710 Axio Observer Z1 microscope</td><td></td></tr><tr><td>Medetomidine (Sedator 1 mg/mL)</td><td>Dechra</td><td>1838 ESP / 020/01/07RFVPT</td><td>Used at a final concentration of 1 mg/kg</td></tr><tr><td>Needle</td><td>BD microlance</td><td>300635</td><td></td></tr><tr><td>No 15 Sterile carbon steel scalpel&lt;br/&gt; Blade</td><td>Swann-Morton</td><td>205</td><td></td></tr><tr><td>Objectives 10x (Plan-Neofluar 10x/0.3), 20x (Plan-Apochromat 20x/0.8) and 40x (Plan-Neofluar 40x/1.30 Oil)</td><td>&amp;nbsp;Carl Zeiss, Germany</td><td></td><td></td></tr><tr><td>Paint brushes</td><td>Van Bleiswijck Amazon B07W7KJQ2X</td><td>&amp;nbsp;Used to handle cryosections</td><td></td></tr><tr><td>Peristaltic pump (Minipuls 3)</td><td>Gilson</td><td>1004170</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS, pH ~ 7.4)</td><td>Sigma-Aldrich, Lyon, France</td><td>P3813</td><td></td></tr><tr><td>Scalpel handle, 125 mm (5&quot;), No. 3</td><td>Swann-Morton</td><td>0208</td><td></td></tr><tr><td>Slide staining system StainTray</td><td>Simport Scientific</td><td>M920</td><td></td></tr><tr><td>Standard diet</td><td>&amp;nbsp;Mucedola</td><td>4RF21</td><td></td></tr><tr><td>Superfrost Plus microscope slides</td><td>Menzel-Glaser, Germany</td><td>J1800AMNZ</td><td></td></tr><tr><td>Tissue-Tek OCT mounting media</td><td>VWR CHEMICALS</td><td>361603E</td><td></td></tr><tr><td>Triglycerides colorimetric assay kit</td><td>Cayman Chemical</td><td>10010303</td><td></td></tr><tr><td>Ultrasonic bath</td><td>Bandelin Sonorex</td><td>&amp;nbsp;TK 52</td><td></td></tr><tr><td>Vannas spring scissors - 3 mm&lt;br/&gt; cutting edge</td><td>Fine Science Tools, Germany</td><td>15000-00</td><td></td></tr><tr><td>ZEN Black software</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

analysis of fluorescent-stained lipid droplets with 3d reconstruction for hepatic steatosis assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

The successful execution of this technique should result in clear lipid droplet staining for the simultaneous characterization of the LD morphology (shape and lipid core density based on the 3D reconstruction) along with their spatial distribution, number per total area, and average size (assessed with the pipeline above described, Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65206/65206fig01.jpg"...

Watch this Scientific Journal Video about Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment at JoVE.com

Author Spotlight: Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

analysis-fluorescent-stained-lipid-droplets-with-3d-reconstruction

Research

JoVE Journal

Biochemistry

5.9K Views.  University of Coimbra. Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Video: Author Spotlight: Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment  

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

Cancer Research

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Medicine

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

This protocol is intended to be a tool to study steatosis and the molecular, biochemical, cellular changes produced by the over exposure of hepatocytes to lipids in vitro.

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

Live Cell Imaging of Lipid Droplet Size and Fusion Using Oil Red O and BODIPY

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

Author Spotlight: Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells  

The present protocol describes how to use oil red O to dye lipid droplets (LDs), calculate the size and number of LDs in a fatty acid-induced fatty hepatocyte model, and use BODIPY 493/503 to observe the process of small LDs fusing into large LDs by live cell imaging.

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety ...

Biology

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Author Spotlight: Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Modeling and Analyzing Retinal Degeneration

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Author Spotlight: Unraveling the Pathogenesis of Age-Related Macular Degeneration and Discovering Potential Therapies 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to ...

Oleic Acid-Induced HepG2 Cells for Metabolic Disease Analysis 

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading to significant health challenges. Previous reports have studied the establishment of animal and cellular models for MASLD, highlighting differences between them. In this study, a cellular model was created by inducing fat accumulation in MASLD. HepG2 cells were stimulated with the unsaturated fatty acid oleic acid at various concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) to emulate MASLD. The model&#39;s efficacy was assessed using cell counting kit-8 assays, Oil Red O staining, and lipid content analysis. This study aimed to create a simple-to-operate cellular model for MASLD cells. Results from the cell counting kit-8 assays showed that the survival of HepG2 cells was dependent on the concentration of oleic acid, with a GI50 of 1.875 mM. Cell viability in the 0.5 mM and 1 mM groups were significantly lower than those in the control group (P &#60; 0.05). Furthermore, Oil Red O staining and lipid content analysis examined fat deposition at varying oleic acid concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) on HepG2 cells. The lipid content of the 0.25 mM, 0.5 mM, and 1 mM groups was significantly higher than that of the control group (P &#60; 0.05). Additionally, triglyceride levels in the OA groups were significantly higher than those in the control group (P &#60; 0.05).

Author Spotlight: Establishing MASLD Cell Models for Investigating Disease Mechanisms and the Lipid-Lowering Effects of Koumiss

This article describes the use of oleic acid-induced HepG2 cells as a model for metabolic dysfunction-associated steatotic liver disease.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

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Chapters in this video

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease