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

Karina Garcia; Andr&#233; Alves; Teresa M. Ribeiro-Rodrigues; Fl&#225;vio Reis; Sofia Viana

doi:10.3791/65206

JoVE Journal > Biochemistry

Biochemistry

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

Published: June 02, 2023

doi:

10.3791/65206

Karina Garcia^1,2,3,4, André Alves^1,2,3, Teresa M. Ribeiro-Rodrigues^1,2,3, Flávio Reis^1,2,3, Sofia Viana^1,2,3,4

¹Institute of Pharmacology & Experimental Therapeutics & Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine,University of Coimbra, ²Center for Innovative Biomedicine and Biotechnology (CIBB),University of Coimbra, ³Clinical Academic Center of Coimbra (CACC), ⁴Polytechnic Institute of Coimbra,Coimbra Health School

Summary

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.

Abstract

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 µm < d ≤ 3 µm), intermediate vesicles (3 µm < d ≤ 9 µm), and macrovesicles (d > 9 µm), 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.

Introduction

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 >1 µm 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 "reference standard" 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&E) analysis, where lipid droplets are evaluated as unstained vacuoles in H&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.

Protocol

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

Pair-house 13 week old male Wistar rats in ventilated cages under controlled environmental conditions of temperature (22 °C ± 1 °C), humidity (50%-60%), and light (12 h light-dark cycle) and with ad libitum access to tap water and standard rodent chow.
After a 2 week period of acclimatization, arbitrarily assign the rats into two groups.
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.
Monitor the body weight (BW) weekly. Record the food and beverage consumption daily per cage.

2. Liver sample preparation

Liver dissection
1. 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 °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).
2. 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.
3. Place the rat on the dissection tray.
4. Sanitize the fur thoroughly with 70% ethanol, and dry it with a paper towel.
5. Using scissors, make a "U"-shaped incision through the skin, and cut open below the diaphragm. Then, cut the rib cage rostrally on the edges to expose the heart.
6. 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.
7. Using scissors and Dumont forceps, remove the liver carefully, and rinse with 1x PBS.
8. Transfer the liver onto a Petri dish, and weigh it.
9. 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 −80 °C for long-term storage.
Liver tissue embedding
1. Prepare a dry ice container, and properly label cryomolds with the sample ID and orientation.
2. Place a few drops of cryo embedding matrix onto the center of the cryomold to make the optimal cutting temperature (OCT).
3. 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.
4. 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.
5. Rapidly place the cryomold with the OCT-covered sample in a dry ice container.
  NOTE: The tissues can be stored at −80 °C for 3 years.

3. Frozen tissue sectioning

Adjust the cryostat temperature (chamber: −21 °C; specimen: −18 °C), and insert a new sterile blade.
Place the samples in the cryostat for 30 min before allowing the temperature to equilibrate.
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.
Place the sample in the specimen head, and trim the surface of the tissue (a few 30 µm tissue sample sections).
Once the region of interest is accessible for sectioning, cut 12 µm 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.
Allow the slides to dry for 10 min at RT.
NOTE: The slides can be stored at −20 °C for 6-12 months or at −80 °C for up to 3 years.

4. BODIPY staining

Thaw the slides in a slide staining system at RT for 30 min.
Wash with 1x PBS (3x for 5 min each).
Circle the section with a hydrophobic layer using a barrier pen.
Prepare 500 µg/mL BODIPY 493/503 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 °C.
NOTE: The solution can be stored at −20 °C for at least 30 days.
Prepare the BODIPY staining solution (1 µg/mL) by adding 2 µL of BODIPY 493/503 stock solution and 0.1 µL of DAPI stock solution (5 mg/mL) to 997.9 µL of 1x PBS.
Incubate the slides (75 µL/slide) with BODIPY 493/503 (1 µg/mL) and DAPI (0.1 µg/mL) at RT for 40 min.
NOTE: Keep the slides in the dark from this step onward.
Wash the slides with 1x PBS (3x for 5 min each).
Mount the slides with coverslips using a fluorescence mounting medium, allow them to dry for 30 min, and seal them with nail polish.
NOTE: The slides can be stored at 4 °C until imaging.

5. Lipid quantification

Measure the serum total cholesterol and TG levels using an automatic, validated method and equipment.
Measure the hepatic TG levels using a triglycerides colorimetric assay kit (Table of Materials) according to the manufacturer's protocol.

6. Image acquisition

Place the slide on the laser scanning confocal microscope slide holder.
For image visualization and acquisition, use a confocal microscope with a 20x objective lens (plan-apochromat: 20x/0.8).
To avoid cross-talk between the BODIPY 493/503 and DAPI, use the sequential (best signal) scan mode on the confocal software.
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.
Use the following settings: pinhole: 1 AU, resolution: 1,024 pixels x 1,024 pixels, bit depth: 12, pixel size: 0.415 µm, bidirectional mode, scan speed: 7 (~1.58 µs/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.
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.
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.
To create wide-area images (Figure 2), change the objective to a10x objective lens (plan-neofluar: 10x/0.3).
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.
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).
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 µm) 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.
Select the Ortho module, and create orthogonal views.
Acquire 3D images by selecting the 3D module following Transparency Rendering Mode with the confocal microscope software.

7. Analysis of images

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.
Click on the Images module in the top-left corner of the CellProfiler window, and upload the images as .tiff files.
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.
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.
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.
To measure the pixel intensity of the identified lipid droplets, add a MeasureObjectIntensity module.
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).
To measure the LDs related to the output data, add the MeasureObjectSizeShape module.
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.
Add an ExportToSpreadsheet module at the end, and click on the Analyze Images button at the bottom-left corner.

8. Statistical analysis

Express the results as mean ± 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.
Analyze the distribution of values using the Kolmogorov-Smirnov test to assess significant deviations from normality. Analyze the parametric data using the Student's unpaired t-test.
NOTE: Values of p < 0.05 were considered statistically significant.

Representative Results

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).

Figure 1: Sample image processing using CellProfiler. (A) Original image of LDs stained with BODIPY 493/503 (green) and nuclei stained with DAPI (blue) in the CTL group. (B) The differentiated LDs (magenta) of the CTL group were overlaid using the OverlayOutlines module. (C) Original image of LDs stained with BODIPY 493/503 (green) and nuclei stained with DAPI (blue) in the HFD group. (D) The differentiated LDs (magenta) of the HFD group were overlaid using the OverlayOutlines module. Images were taken at 20x magnification using a laser point scanning confocal microscope. Scale bars = 50 µm. Abbreviations: CTL = control group; HFD = high-fat diet group. Please click here to view a larger version of this figure.

Serum TGs, total cholesterol, LDL-c, HDL-c, as well as the hepatic TG contents were evaluated (Table 1). HFD-fed animals presented a pronounced dyslipidemic profile, characterized by an accentuated accumulation of hepatic TGs (345% when compared to CTL, p < 0.001), along with a subtle increase in circulating TG levels (129% when compared to CTL, p > 0.05).

	Parameter	CTL	HFD
Serum
	Total Cholesterol (mg/dL)	56.67 ± 9.35	80.40 ± 7.45
	LDL (mg/dL)	7.66 ± 0.84	7.40 ± 1.03
	HDL (mg/dL)	17.83 ± 2.93	28.40 ± 2.40 *
	Triglycerides (mg/dL)	154.8 ± 40.17	201.2 ± 38.12
Liver
	Triglycerides (mg/g)	15.29 ± 1.31	52.83 ± 6.73 ***

Table 1: Serum TGs, total cholesterol, LDL-c, HDL-c, and the hepatic TG contents. Data are expressed as mean ± SEM (n = 5-6 per group). *** p < 0.001 versus CTL. Statistical analysis was performed using a Student's unpaired t-test. Abbreviations: CTL = control group; HFD = high-fat diet group.

Figure 2 shows representative wide-area images (tile scans with a 10x objective) of liver sections in which LDs are stained with BODIPY 493/503 (green) and nuclei are stained with DAPI (blue). Individual LDs of various sizes were successfully visualized with BODIPY 493/503 staining, which showed a widespread distribution pattern in the HFD-fed animals.

Figure 2: Representative images of LD accumulation in hepatic tissue. LDs stained with BODIPY 493/503 (green), and nuclei stained with DAPI (blue). Images were taken at 10x magnification using a laser point scanning confocal microscope. Scale bars = 200 µm. Abbreviations: CTL = control group; HFD = high-fat diet group. Please click here to view a larger version of this figure.

Figure 3A shows representative orthogonal projections with a 20x objective and 3D images with a 40x objective of hepatic LDs. Single-plane images were processed and analyzed by CellProfiler (version 4.2.5) to assess the fluorescence intensity, number, area, and diameter of the LCs (Figure 3B–E). It was possible to confirm that the HFD-fed animals displayed an increased number of hepatic LDs (151% vs. CTL, Figure 3B), and this was corroborated by the augmented BODIPY 493/503 fluorescence intensity (182% vs. CTL, p < 0.001, Figure 3C). Moreover, the area ratio of LDs nearly tripled (360% vs. CTL, p < 0.0001, Figure 3D) as they presented larger diameters (182% vs. CTL, Figure 3E) in the HFD-fed animals. To assess the size distribution, the LDs were categorized into three groups according to the diameter ranges: 1 µm < d ≤ 3 µm, 3 µm < d ≤ 9 µm, and d > 9 µm (Figure 3F). The animals fed the HFD showed a greater than 20% increase in the number of supersized, macrovesicular hepatic LDs (d > 9 µm, p < 0.0001) along with a nearly three-fold reduction in the number of microvesicles smaller than 3 µm in diameter (1 µm < d ≤ 3 µm, p < 0.0001).

Figure 3: Orthogonal/3D views of LDs and data analysis. (A) Representative orthogonal projections and 3D rendered images of hepatic lipid droplets (green, BODIPY 493/503) and nuclei (blue, DAPI) in the liver of mice fed a normal or HFD diet. Images were taken at 20x (left) and 40x (right) magnification using a laser point scanning confocal microscope. Scale bars = 20 µm. (B) Number of hepatic LDs. (C) Fluorescence intensity of the hepatic LDs. (D) Area ratio of the hepatic LDs. (E) Lipid droplet diameters. (F) Fractional ratios of groups of hepatic lipid droplets with different diameters: 1 µm < d ≤ 3 µm, 3 µm < d ≤ 9 µm, and d > 9 µm. Data are presented as mean ± SEM. n = 5-6 mice/group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical analysis was performed using a Student's unpaired t-test. Abbreviations: CTL = control group; HFD = high-fat diet group. Please click here to view a larger version of this figure.

Discussion

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. Subsequently, the degree of LD accumulation was visualized by the fluorescent probe BODIPY 493/503 under low magnification. As expected, the BODIPY 493/503 staining revealed a widespread distribution of vesicular structures across the hepatic tissue in the HFD group. Resorting to orthogonal projections and 3D reconstructions, it was possible to observe that the LD core presented a full content of neutral lipids, which appeared as nearly spherical droplets. Moreover, the robust increase in the LD area per total area was clearly evident (360% vs. CTL group), and this area was most likely filled with neutral TGs given their quantitative score in the HFD group (52.83 mg/g ± 6.73 mg/g; 346% vs. CTL group). To further characterize the LD dynamics upon HFD feeding, the micro or macrovesicular nature of hepatic steatosis was analyzed. Using the LD diameter ranges previously described in literature⁴^,⁴⁶^,⁴⁷, it was possible to discriminate a significant drop in the microvesicular LD count (1 µm < d ≤ 3 µm), which paralleled a proportional increase in LD macrovesicles (d > 9 µm). Several studies have reported that LDs in hepatocytes can form supersized LDs (up to tens of microns in diameter)⁴^,⁴⁶^,⁴⁷, which is in line with the present results.

In recent years, classical lipid dyes have been gradually substituted with a new array of fluorescent lipophilic probes, such as BODIPY, given their neutral and planar structure stabilized by a difluoroboron complex⁴⁸; these probes have been shown to be very effective at tagging LDs to study their morphology, dynamics, and interaction with other organelles in living cells and some fixed tissues⁴⁹^,⁵⁰. Within the fluorescent-stained lipid droplets protocol presented herein for hepatic steatosis assessment, there are some critical steps for the success of the technique that mostly rely on tissue preparation and image acquisition. Since the BODIPY signal can be bleached by UV light⁵¹, LD imaging acquisition must occur before imaging other fluorescent dyes that are excited by UV light, such as the commonly known DNA fluorescent dyes. Furthermore, it is highly recommended to protect the microscope slides from light exposure in order to prevent BODIPY fluorescence quenching before imaging. Even though the 3D reconstruction of LDs is very useful for the study of their morphology, it is extremely time-consuming, since 3D-reconstructed images are composed of several independent 2D images. Therefore, researchers should consider avoiding this step when the primary goal of the experiment is solely the assessment of micro and macrovesicular hepatic steatosis. To avoid bias, two independent observers blinded to the treatment group should carry out the data acquisition and analysis. The semi-automated image processing software (Cell Profiler pipeline) further contributes to a semi-blind quantification and overcomes the increased processing time consistently observed in manual analysis.

This technique may be extended to broader applications in combination with immunohistochemistry staining of other tissues and species, as long as the optimal BODIPY concentrations and image acquisition settings have been determined to maximize the signal/background ratio. Such experimental settings may allow the simultaneous detection of other antigens provided that the second fluorophore used does not overlap with the BODIPY excitation/emission spectra.

Overall, the optimized BODIPY 493/503 fluorescence-based protocol presented herein is a reliable and simple tool for LD characterization and may represent a complementary approach to the classical histological protocols often employed to confirm and grade hepatic steatosis.

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Garcia, K., Alves, A., Ribeiro-Rodrigues, T. M., Reis, F., Viana, S. Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment. J. Vis. Exp. (196), e65206, doi:10.3791/65206 (2023).

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