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

3D Fluorescence Lipid Droplet Reconstruction을 통한 간 지방증 평가

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.

저자 스포트라이트: 간 지방증 평가를 위한 3D 재구성을 통한 형광 염색 지질 방울 분석  

간 지방증 평가를 위한 3D 재구성을 통한 형광 염색 지질 방울 분석

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  

지질 방울의 이미지 획득 및 분석

레이저 스캐닝 컨포칼 현미경을 켜고 슬라이드를 슬라이드 홀더에 놓습니다. 이미지 시각화 및 획득을 위해 20X 대물 렌즈를 선택합니다. 컨포칼 소프트웨어에서 순차 스캔 모드를 선택하여 BODIPY 493/503과 DAPI 간의 혼선을 방지합니다.그런 다음 488 나노미터 아르곤 레이저 라인을 사용하여 BODIPY 493/503 다이를 자극하고 405 나노미터 레이저 라인을 사용하여 DAPI 염색을 자극합니다. 방출 범위를 BODIPY의 경우 493 - 589 나노미터, DAPI의 경우 410 - 464 나노미터로 설정합니다. 그런 다음 프레임 크기, 스캔 속도 및 평균을 조정하여 12비트의 2배 비트 깊이, 양방향의 방향, 스캔 영역 디지털 줌에서 1을 뺀 값, 핀홀에서 1을 뺀 영역 단위를 포함하는 평균을 구성합니다.게인 및 디지털 게인 설정을 조정합니다. 범위 표시기에 표시된 대로 포화 픽셀이 없는지 확인하십시오. 지질 방울을 정확하게 식별한 후 BODIPY 및 DAPI 채널로 이미지를 획득합니다.광역 이미지를 만들려면 10X 주관 렌즈로 전환하십시오. 그런 다음 타일 스캔 모드를 활성화하고 5x5 모자이크 이미지를 생성하도록 구성합니다. 3D 및 직교 뷰를 생성하려면 커버 유리 상단에 이멀젼 오일 한 방울을 바르고 대물 렌즈를 40X 렌즈로 변경합니다.Z 스택 모드를 선택합니다. Z 평면을 조정하여 이미지 캡처를 위한 첫 번째 및 마지막 위치를 정의하고 초점이 맞춰진 모든 물방울을 캡처하기 위해 약 0.5미크론의 광학 슬라이스 두께를 보장합니다. 직교 모듈을 선택하고 직교 뷰를 생성합니다.투명 렌더링 모드에 따라 3D 모듈을 선택하여 3D 이미지를 수집합니다. 영상 획득 후 CellProfiler를 사용하여 단면 영상 처리 및 분석을 시작합니다. CellProfiler 인터페이스의 왼쪽 상단 모서리에 있는 이미지 모드를 클릭하여 TIF 형식의 이미지를 업로드합니다.names and types 모듈을 사용하여 파일 이름을 기준으로 BODIPY 방울과 DAPI 핵 염색 이미지를 정렬합니다. 지질 액적 분석의 경우 BODIPY 염색 이미지만 사용합니다. 모듈 조정을 클릭하여 파이프라인 구성을 시작하고 색상을 회색으로 모듈 선택하여 이미지를 그레이 스케일로 변환합니다.그런 다음 기본 객체 식별 모듈을 사용하여 6에서 300 픽셀 사이의 지질 방울과 1의 임계 보정 계수를 정의하여 그레이 스케일 이미지 내에서 지질 방울을 감지합니다. 식별된 지질 방울의 픽셀 강도를 측정하려면 측정 객체 강도 모듈을 추가합니다. 추가 필터 개체 모듈을 통합하여 가장 강한 신호만 정량화하고 덜 강한 신호는 최종 지질 액적 분석에서 제외하도록 합니다.다음으로, 출력 데이터와 관련된 지질 방울을 측정하려면 측정 개체 크기 형상 모듈을 추가합니다. 그런 다음 오버레이 윤곽선 모듈을 추가하여 식별된 물방울을 원본 이미지에 오버레이하여 분할이 처리되지 않은 이미지에서도 정확하게 보이도록 합니다. 완료되면 스프레드시트로 내보내기 모듈을 추가하고 왼쪽 하단 모서리에서 이미지 분석 버튼을 클릭합니다.대조군과 비교하여 HFD를 먹인 동물은 뚜렷한 이상지질혈증 프로필과 순환 트리글리세리드 수치의 미묘한 증가를 나타냈습니다. 광역 이미지는 HFD를 먹인 동물의 간에서 증가된 지질 방울을 보여주었습니다. CellProfiler를 사용한 분석 결과 간 지질 방울의 수가 증가하고, BODIPY 형광 강도가 증가하고, 면적 비율이 360%이며, 직경이 더 커진 것으로 나타났습니다.HFD를 먹인 동물의 크기 분포는 9 미크론 이상의 거대 소포 간 지질 방울의 증가와 3 미크론 미만의 미세 소포의 감소를 나타냈다.

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 ...

연구

생화학

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.

냉동 간 조직 절편 및 지질 방울의 BODIPY 염색

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.