Name: Image Acquisition of Lipid Droplets and Analysis
Uploaded: 2023-06-02T12:40:00
Description: Watch this Scientific Journal Video about Image Acquisition of Lipid Droplets and Analysis at JoVE.com

The video demonstrates the procedure for using a laser scanning confocal microscope to visualize and analyze lipid droplets in biological samples. It outlines settings adjustment, image acquisition, and post-processing with CellProfiler to evaluate lipid droplet characteristics, ultimately comparing results between control and high-fat diet (HFD) groups.

image acquisition of lipid droplets and analysis

使用 3D 荧光脂滴重建评估肝脂肪变性

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.

作者聚焦： 使用 3D 重建分析荧光染色脂滴用于肝脂肪变性评估  

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

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.

Watch this Scientific Journal Video about Image Acquisition of Lipid Droplets and Analysis at JoVE.com

Image Acquisition of Lipid Droplets and Analysis  

脂滴的图像采集和分析

打开激光扫描共聚焦显微镜并将载玻片放在载玻片支架上。对于图像可视化和采集，请选择20X物镜。从共聚焦软件中，选择顺序扫描模式以避免 BODIPY 493/503 和 DAPI 之间的串扰。然后使用 488 纳米氩激光线激发 BODIPY 493/503 模具，使用 405 纳米激光线激发 DAPI 染色。将 BODIPY 的发射范围设置为 493 至 589 纳米，将 DAPI 的发射范围设置为 410 至 464 纳米。接下来，通过调整帧尺寸、扫描速度和平均值来配置显微镜设置，其中包括数字两倍位深度为 12 位、方向为双向、扫描区域数字变焦减 1 和针孔减去一个区域单位。调整增益和数字增益设置。确保没有范围指示器指示的饱和像素。准确识别脂滴后，使用BODIPY和DAPI通道获取图像。要创建广域图像，请切换到 10 倍主观镜头。然后激活平铺扫描模式并将其配置为生成五乘五的马赛克图像。要生成 3D 和正交视图，请在盖玻片顶部涂抹一滴浸油，然后将物镜更换为 40X 镜头。选择 Z 堆栈模式。调整 Z 平面以定义图像捕获的第一个和最后一个位置，确保光学切片厚度约为 0.5 微米，以捕获聚焦的所有液滴。选择正交模块并创建正交视图。通过按照透明渲染模式选择3D模块来获取3D图像。图像采集后，开始使用CellProfiler处理和分析单平面图像。通过单击CellProfiler界面左上角的图像模式，以TIF格式上传图像。使用名称和类型模块根据文件名在 BODIPY 液滴和 DAPI 细胞核染色图像之间进行排序。对于脂滴分析，仅使用BODIPY染色图像。通过单击调整模块启动管道构建，并选择颜色为灰度模块以将图像转换为灰度。然后使用识别主要对象模块，定义6至300像素之间的脂质液滴和1的阈值校正因子，以检测灰度图像内的脂滴。要测量已识别的脂滴的像素强度，请添加测量对象强度模块。结合一个额外的过滤器对象模块，以确保只有最强的信号被量化，而强度较低的信号被排除在最终的脂滴分析之外。接下来，要测量与输出数据相关的脂滴，请添加测量对象大小形状模块。然后添加一个叠加轮廓模块，将识别出的液滴叠加在原始图像上，从而确保分割在未处理的图像上看起来也很准确。完成后，添加导出到电子表格模块，然后单击左下角的分析图像按钮。与对照组相比，HFD喂养的动物表现出明显的血脂异常特征和循环甘油三酯水平的微妙增加。广域图像显示HFD喂养动物肝脏中的脂滴增加。使用CellProfiler分析显示，肝脂液滴数量增加，BODIPY荧光强度增强，面积比360%和直径更大。HFD喂养动物的尺寸分布显示，大于9微米的大囊泡肝脂滴增加，小于3微米的微囊泡减少。

image-acquisition-of-lipid-droplets-and-analysis

Research

JoVE Journal

Biochemistry

Image Acquisition of Lipid Droplets and Analysis

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.

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 ...

科研

生物化学

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.

脂滴的冷冻肝组织切片和BODIPY染色

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.