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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;,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 
			and flammable. Vapors may cause 
			irritation. Manipulate in a fume 
			hood. Avoid direct contact with skin. 
			Wear rubber gloves, protective eye 
			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, &#248; 
			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&#160; 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&#160; (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 
			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>&#160;Carl Zeiss, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brushes</td>
			<td>Van Bleiswijck Amazon B07W7KJQ2X</td>
			<td>&#160;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&#34;), 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>&#160;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>&#160;TK 52</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas spring scissors - 3 mm 
			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.

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

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

This imaging approach allows the three-dimensional reconstruction of hepatic lipid droplet deposition and overcomes the main disadvantages of classic histological staining that narrowed the successful discrimination between micro and macrovesicular steatosis. The assessment of microvesicular steatosis is when gold-standard histological stainings are used, both and Oil Red, due to disruption and occasional fusion of native lipid droplets. The pro-BODIPY offers an alternative to the routine protocols for microvesicular hepatic steatosis assessment due to its right hydrophobicity for a fast uptake and effective tagging of lipid droplets, being not only insensitive to the environment polarity, but also extremely selective.This imaging approach encloses broader applications in combination with immunochemistry techniques, allowing the simultaneous detection of other subcellular antigens that may advance the study of lipid droplets'biogenesis and dynamics, underpinning micro and macrovesicular hepatic steatosis.

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

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.

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

Frozen Liver Tissue Sectioning and BODIPY Staining of Lipid Droplets

Turn on the cryostat and cut 12-micron-thick sections of the frozen liver tissue collected from a rat. Place the sections onto the labeled microscope slides at room temperature. For BODIPY staining, wash the tissue sections thrice with PBS for five minutes each.After the last wash, circle the sections on the slide with a hydrophobic layer using a barrier pen. Prepare a staining solution containing BODIPY 493/503 and DAPI. Then, pipette 75 microliters of the solution onto each slide.Incubate the slides at room temperature for 40 minutes in the dark. Following incubation, wash the slides thrice with PBS for five minutes each. Then, using a fluorescence mounting medium, carefully mount cover slips on each slide.Allow the slides to dry for 30 minutes before sealing the edges with nail polish.

Image Acquisition of Lipid Droplets and Analysis

Turn on the laser scanning confocal microscope and place the slide on the slide holder. For image visualization and acquisition, select the 20X objective lens. From the confocal software, select sequential scan mode to avoid crosstalk between the BODIPY 493/503 and DAPI.Then excite the BODIPY 493/503 die using the 488 nanometer argon laser line and the DAPI stain using the 405 nanometer laser line. Set the emission ranges at 493 to 589 nanometers for BODIPY and 410 to 464 nanometers for DAPI. Next, configure the microscope settings by adjusting frame sizes, scan speed, and averaging which includes number two times bit depth as 12 bits, direction as bidirectional, scan area digital zoom minus one, and pinhole minus one area unit.Adjust the gain and digital gain settings. Ensure there are no saturated pixels as indicated by the range indicator. After accurately identifying the lipid droplets, acquire the image with the BODIPY and DAPI channels.To create wide area images, switch to a 10X subjective lens. Then activate tile scan mode and configure it to generate five by five mosaic images. To generate 3D and orthogonal views, apply a drop of immersion oil on the top of the cover glass and change the objective lens to a 40X lens.Select the Z-stack mode. Adjust the Z-plane to define the first and last positions for image capture, ensuring an optical slice thickness of approximately 0.5 microns to capture all droplets in focus. Select the ortho module and create orthogonal views.Acquire 3D images by selecting the 3D module following transparency rendering mode. After image acquisition, begin processing and analyzing single plane images using CellProfiler. Upload the images in TIF format by clicking on the images mode in the top left corner of the CellProfiler interface.Use the names and types module to sort between BODIPY droplet and DAPI nuclei stained images based on the filenames. For lipid droplet analysis, use only BODIPY stained images. Initiate pipeline construction by clicking on adjust modules and select the color to gray module to convert the images into gray scale.Then using identify primary objects module, define lipid droplets between 6 to 300 pixels and a threshold correction factor of one to detect lipid droplets within the gray scale images. To measure the pixel intensity of the identified lipid droplets, add a measure object intensity module. Incorporate an additional filter objects module to ensure that only the strongest signals are quantified while less intense signals are excluded from the final lipid droplet analysis.Next, to measure the lipid droplets related to the output data, add the measure object size shape module. Then add an overlay outlines module to overlay the identified droplet on the original image and thus ensure that the segmentation looks accurate on the unprocessed image as well. Once done, add an export to spreadsheet module and click on the analyze images button from the bottom left corner.Compared to the control, HFD-fed animals presented a pronounced dyslipidemic profile and subtle increase in circulating triglycerides levels. Wide area images revealed increased lipid droplets across the livers of HFD-fed animals. Analysis with CellProfiler revealed an increased number of hepatic lipid droplets, augmented BODIPY fluorescence intensity, 360%area ratio, and larger diameters.Size distribution in HFD-fed animals revealed an increase in macro vesicular hepatic lipid droplets of more than nine microns and a reduction in microvesicles of less than three microns.

2.8K 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.

Assessing Hepatic Steatosis with 3D Fluorescence Lipid Droplet Reconstruction

Summary