这项研究由国家和欧洲基金 通过 葡萄牙科学技术基金会（FCT），欧洲区域发展基金（FEDER）和竞争运营因素计划（COMPETE）：2020.09481.BD，UIDP / 04539 / 2020（CIBB）和POCI-01-0145-FEDER-007440资助。作者要感谢iLAB - 显微镜和生物成像实验室的支持，科英布拉大学医学院的设施，国家基础设施PPBI-葡萄牙生物成像平台（POCI-01-0145-FEDER-022122）的成员，以及FSE CENTRO-04-3559-FSE-000142的支持。

本研究中执行的所有动物程序均已获得科英布拉临床和生物医学研究所（iCBR）动物福利机构（ORBEA，#9 / 2018）的批准，并符合动物护理国家和欧洲指令以及ARRIVE指南。
1. 实验设计
<ol><li>在温度（22°C±1°C），湿度（50%-60%）和光照（12小时明暗循环）的受控环境条件下，在通风笼中配对13周龄雄性Wistar大鼠，并 随意 获得自来水和标准啮齿动物食物。</li>
	<li>在适应2周后，将大鼠任意分为两组。</li>
	<li>分别用标准食物和高脂肪饮食（45% kcal/脂肪）喂养对照组（CTRL，n = 6）和高脂肪饮食组（HFD，n = 6），持续 24 周。</li>
	<li>每周监测体重（BW）。记录每个笼子每天的食物和饮料消耗量。</li>
</ol>2. 肝脏样本制备
<ol><li>肝脏清扫<ol><li>准备蠕动泵：通过管路运行70%乙醇，将27 G针头连接到管子的出口端，并用冰冷（4°C）1x磷酸盐缓冲盐水（PBS，pH ~7.4）灌注管子（例如，在蠕动泵上200 rpm，内径为1.6毫米硅管，IV迷你滴注组）。调整体重（例如，对于100-150g大鼠，使用约10-12mL / min的流速）。</li>
		<li>使用麻醉方案通过腹膜内注射麻醉大鼠（氯胺酮的终浓度= 75mg / kg，美托咪定的最终浓度= 1mg / kg）。 注意：确保使用脚趾捏反应方法完全麻醉大鼠。</li>
		<li>将大鼠放在解剖盘上。</li>
		<li>用70%乙醇彻底消毒毛皮，然后用纸巾擦干。</li>
		<li>用剪刀在皮肤上做一个“U”形切口，然后在横膈膜下方切开。然后，在边缘切开肋骨，露出心脏。</li>
		<li>当 1x PBS 运行时，将针头穿过左心室进入升主动脉，然后夹紧。然后，切开右心房，以便在灌注开始后进行引流。 注意：当血液被PBS取代时，肝脏应该开始变白。</li>
		<li>使用剪刀和杜蒙镊子，小心取出肝脏，并用 1x PBS 冲洗。</li>
		<li>将肝脏转移到培养皿上，称重。</li>
		<li>使用手术刀，从肝脏左叶的侧侧（距边缘 1 厘米）收集 5 毫米厚的肝组织样本以进行包埋过程。 注意剩余的组织可以储存在-80°C以长期储存。</li>
	</ol></li>
	<li>肝组织包埋<ol><li>准备一个干冰容器，并在冷冻模具上正确贴上样品ID和方向标签。</li>
		<li>将几滴冷冻包埋基质滴在冷冻机的中心，以获得最佳切割温度（OCT）。</li>
		<li>确保组织样品的方向正确适合横截面。 注意：确保接触冷冻机底部的一侧是将首先切片的一侧。</li>
		<li>小心地将更多的OCT滴在冷冻机上，直到它完全被覆盖。尽量避免形成气泡。如有必要，用普通镊子去除OCT内的任何气泡。</li>
		<li>将带有OCT覆盖的样品的冷冻快速放入干冰容器中。 注意：组织可以在-80°C下储存3年。</li>
	</ol></li>
</ol>3. 冷冻组织切片
<ol><li>调节低温恒温器温度（腔室：-21°C;试样：-18°C），并插入新的无菌刀片。</li>
	<li>将样品放入低温恒温器中30分钟，然后让温度平衡。</li>
	<li>在试样盘上制作单层OCT，以允许样品粘附。让OCT冷冻，并将组织样品安装在所需的方向。 注意： 切割表面应与刀片平行。为了保持良好的附着力，请将热提取器放在样品的顶部。前面的步骤也可以在装有干冰的干冰容器中进行。</li>
	<li>将样品放在标本头中，并修剪组织表面（几个30μm组织样品切片）。</li>
	<li>一旦感兴趣区域可以切片，切割12μm厚的切片，并将它们放在室温（RT）下保存的标记显微镜载玻片上。 注意：要收集切片，请慢慢将载玻片移向组织切片。每张载玻片可以收集两个肝脏切片。</li>
	<li>让载玻片在室温下干燥10分钟。 注意：载玻片可在-20°C下储存6-12个月或在-80°C下储存长达3年。</li>
</ol>4. 胸部染色
<ol><li>在室温下在载玻片染色系统中解冻载玻片30分钟。</li>
	<li>用 1 次 PBS（每次 3 次，每次 5 分钟）洗涤。</li>
	<li>使用屏障笔用疏水层圈出该部分。</li>
	<li>准备 500 μg/mL BODIPY 493/503 储备溶液：将 1 mg BODIPY 493/503 溶解在 2 mL 溶剂（90% DMSO;10% 1x PBS）中。避光。在37°C的超声波浴中超声处理1小时（9.5-10W）。 注意：溶液可以在-20°C下储存至少30天。</li>
	<li>通过将 2 μL BODIPY 493/503 储备溶液和 0.1 μL DAPI 储备溶液 （5 mg/mL） 加入 997.9 μL 1x PBS 中来制备 BODIPY 染色溶液 （1 μg/mL）。</li>
	<li>将载玻片（75 μL/载玻片）与 BODIPY 493/503 （1 μg/mL） 和 DAPI （0.1 μg/mL） 在室温下孵育 40 分钟。 注意：从此步骤开始，将幻灯片保持在黑暗中。</li>
	<li>用 1x PBS（每次 3 次，每次 5 分钟）清洗载玻片。</li>
	<li>使用荧光封片介质用盖玻片安装载玻片，让它们干燥30分钟，并用指甲油密封。 注意：载玻片可以储存在4°C直至成像。</li>
</ol>5. 脂质定量
<ol><li>使用经过验证的自动方法和设备测量血清总胆固醇和TG水平。</li>
	<li>根据制造商的方案，使用甘油三酯比色测定试剂盒（材料表）测量肝脏TG水平。</li>
</ol>6. 图像采集
<ol><li>将载玻片放在激光扫描共聚焦显微镜载玻片支架上。</li>
	<li>对于图像可视化和采集，请使用带有20倍物镜的共聚焦显微镜（平面复消色差：20x/0.8）。</li>
	<li>为避免 BODIPY 493/503 和 DAPI 之间的串扰，请在共聚焦软件上使用顺序（最佳信号）扫描模式。</li>
	<li>使用 488 nm 氩激光线激发 BODIPY 593/503，使用 405 nm 激光线激发 DAPI。将 BODIPY 493/503 的发射范围设置为 493-589 nm，为 DAPI 设置 410-464 nm。</li>
	<li>使用以下设置：针孔：1 AU，分辨率：1，024 像素 x 1，024 像素，位深度：12，像素大小：0.415 μm，双向模式，扫描速度：7（20 倍物镜为 ~1.58 μs/像素），线平均：2 倍，数码变焦：1。 注意：上述扫描参数必须针对所使用的每个共聚焦显微镜和物镜进行优化。</li>
	<li>适当设置增益和数字增益，以便在范围指示器上检测到饱和像素。 注意：通过调整偏移来校正背景信号。</li>
	<li>正确识别LD后，使用BODIPY和DAPI通道获取图像。 注：每个颜色通道的所有照片必须在相同的条件下（曝光和常规设置）拍摄。</li>
	<li>要创建广域图像（图2），请将物镜更改为a10x物镜（plan-neofluar：10x/0.3）。</li>
	<li>选择 平铺扫描 模式，每 5 张图像制作 5 个马赛克。 注意：在本工作中，每个图块都有 10% 的重叠，以便适当地合并图块进行分析。</li>
	<li>要创建3D和正交视图（图3A），请将一滴浸油放在盖玻片的顶部，并将物镜更改为40倍物镜（Plan-neofluar：40x/1.30油）。</li>
	<li>选择 Z-Stack 模式，并通过调整Z平面，用最佳厚度（~0.5μm）的光学切片定义采集的第一个和最后一个位置，以确保捕获所有液滴。 注意：为了加快图像采集速度并避免漂白，可以将光学切片调整到次优尺寸，确保至少 30% 的过采样以实现良好的 3D 重建。</li>
	<li>选择正 交 模块，然后创建正交视图。</li>
	<li>通过使用共聚焦显微镜软件在透明渲染模式下选择3D模块来获取3D图像。</li>
</ol>7. 图像分析
<ol><li>使用CellProfiler（版本4.2.5）处理和分析单平面图像（20倍放大倍率）。 注意：这项工作中使用的管道改编自Adomshick等人。45.</li>
	<li>单击CellProfiler窗口左上角 的图像模块， 然后将图像作为.tiff文件上传。</li>
	<li>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。仅使用BODIPY染色图像进行LD分析。</li>
	<li>要启动管道构造，请单击调整 模块，然后选择模块 ColorToGray 以将图像转换为灰度图像。 注意：要识别对象，需要灰度图像。</li>
	<li>通过使用灰度图像的 IdentifyPrimaryObjects 模块来识别液滴。 注意：必须调整此模块中的参数才能完成良好的LD识别。在这项工作中，定义大小在6像素到300像素之间且阈值校正因子为1.0的LD导致了最准确的识别。</li>
	<li>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。</li>
	<li>添加一个额外的 FilterObjects 模块，以确保仅量化最强的信号，而从最终的脂滴分析中排除强度较低的信号（最小强度：0.15;最大强度：1 任意单位）。</li>
	<li>若要测量与输出数据相关的 LD，请添加 MeasureObjectSizeShape 模块。</li>
	<li>此时添加一个 OverlayOutline 模块，将识别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准确。 注意：这是一个可选的（质量控制）步骤。</li>
	<li>在末尾添加一个 导出到电子表格 模块，然后单击左下角的 “分析图像 ”按钮。</li>
</ol>8. 统计分析
<ol><li>使用任何统计分析软件应用程序将结果表示为平均值（S.E.M.）的平均值±标准误差。 注意：在本研究中，GraphPad软件用于统计分析。</li>
	<li>使用柯尔莫哥罗夫-斯米尔诺夫检验分析值的分布，以评估与正态性的显著偏差。使用学生非配对 t 检验分析参数数据。 注： p < 0.05的值被认为具有统计学意义。</li>
</ol>

使用 3D 荧光脂滴重建评估肝脂肪变性

作者没有什么可透露的。

这种基于荧光的 BODIPY 493/503 LD 评估方案旨在开发一种用于评估肝脂肪变性的新型成像方法。鉴于肥胖与脂肪肝之间的强相关性，采用西式高脂肪饮食建立肝脂肪变性动物模型26。定量甘油三酯比色测定试剂盒证实了肝TG含量的强劲增加，这表明HFD喂养的动物的肝脏脂质沉积情况增加。随后，在低放大倍率下，用荧光探针BODIPY 493/503观察LD积累的程度。正如预期的那样，BODIPY 493/503 ?...

脂滴（LDs），通常被视为能量库，是介导脂质储存的特化细胞器，它们包括疏水性中性脂质核心，主要含有胆固醇酯和甘油三酯（TG），由磷脂单层封装1，2，3。
LD生物发生发生在内质网（ER）中，从三酰基甘油（TAG）和甾醇酯的合成开始。中性脂质以低浓度在ER双层的小叶之间扩散，但聚结成油状体，当其细胞内浓度增加时，油状体从ER膜生长并芽成近球形液滴4。随后，来自ER双层和细胞质的蛋白质，特别是脂周（PLIN）蛋白家族，易位到LD的表面以促进出芽5，6，7，8，9。
通过新的脂肪酸合成和LD融合或聚结，LD长成不同的大小。因此，不同细胞类型中LD的大小和数量差异很大。小液滴（直径300-800nm），被称为初始LD（iLDs），几乎可以由所有细胞形成4。在LD形成的后期，大多数细胞能够将一些iLD转化为更大的iLDs-膨胀LDs（eLDs直径>1μm）。然而，只有特定的细胞类型，如脂肪细胞和肝细胞，才有能力形成巨大或超大的LD（直径可达数十微米）4，10。
LDs在调节细胞脂质代谢，抑制脂毒性，预防ER应激，线粒体功能障碍以及最终由游离脂肪酸（FAs）引起的细胞死亡方面起着非常重要的作用11，12，13，14。此外，LDs还与基因表达，病毒复制蛋白隔离以及膜运输和信号传导的调节有关15，16，17。因此，LD生物发生的失调是与代谢综合征，肥胖，2型糖尿病（T2DM）和/或动脉硬化相关的慢性疾病的标志，仅举几例18，19，20。
肝脏作为代谢中枢，主要负责通过储存和处理脂质的脂质代谢，因此，它不断受到脂毒性的威胁21。肝脂肪变性（HS）是一系列进行性肝病的共同特征，其特征是细胞内脂质以胞质LD的形式过度积累，最终可能导致肝脏代谢功能障碍，炎症和晚期非酒精性脂肪性肝病22，23，24，25.当极低密度脂蛋白（VLDL）中脂肪酸氧化和作为甘油三酯输出的速率低于血浆和从头脂肪酸合成中摄取肝脂肪酸的速率时，就会发生HS26。脂质的肝脏蓄积通常以两种形式发生 - 微泡和大泡脂肪变性 - 并且它们显示出不同的细胞结构特征27。通常，微泡性脂肪变性的特征是存在分散在整个肝细胞中的小LD，细胞核位于中央，而大泡性脂肪变性的特征是存在单个大LD，该LD占据肝细胞的大部分，将细胞核推向外围28，29.值得注意的是，这两种类型的脂肪变性经常一起发现，目前尚不清楚这两种LD模式如何影响疾病发病机制，因为证据仍然不一致31，32，33，34。然而，这种类型的分析通常被用作临床前和临床研究中的“参考标准”，以了解LD的动态行为并表征肝脂肪变性29，34，35，36。
肝活检是诊断和分级HS的金标准，通过组织学苏木精和伊红（H&E）分析常规评估，其中脂质液滴被评估为H&E染色肝脏切片中的未染色液泡37。虽然可以接受大泡脂肪变性评估，但这种类型的染色通常会缩小微泡脂肪变性的评估范围38。脂溶性重氮染料，如油红O（ORO），通常与明场显微镜结合使用以分析细胞内脂质储存，但这些仍然有许多缺点：（i）在染色过程中使用乙醇或异丙醇，尽管细胞是固定的，但这通常会导致天然LD的破坏和偶尔的融合39;（ii）耗时，因为ORO溶液由于保质期有限而需要新鲜粉末溶解和过滤，从而导致结果不太一致;（iii）以及ORO不仅染色脂滴，而且经常高估肝脂肪变性的事实38。
因此，细胞渗透性亲脂性荧光团（如尼罗红）已被用于活样品或固定样品中，以克服上述一些限制。然而，细胞脂质细胞器标记的非特异性性质反复缩小LD评估的范围40。此外，尼罗河红的光谱特性根据环境的极性而变化，这通常会导致光谱偏移41。
亲脂性荧光探针1，3，5，7，8-五甲基-4-硼-3a，4adiaza-s-茚达烯（激发波长：480nm;最大发射波长：515nm;BODIPY 493/503）表现出疏水性特性，允许其被细胞内LD快速吸收，积聚在脂滴核心中，随后发出亮绿色荧光12。与尼罗红不同，BODIPY 493/503对环境极性不敏感，并且已被证明具有更高的选择性，因为它在LD成像中显示出高亮度。为了染色中性LD，该染料可用于活细胞或固定细胞，并成功与其他染色和/或标记方法偶联42。染料的另一个优点是它几乎不需要努力地放入溶液中并且是稳定的，因此无需为每个实验新鲜制备42。尽管BODIPY 493/503探针已成功用于可视化细胞培养物中LD的定位和动力学，但一些报告也证明了这种染料在组织中的LD成像工具的可靠使用，包括人股外侧肌43，大鼠比目鱼肌42和小鼠肠道44。
在本文中，我们提出了一种优化的基于 BODIPY 493/503 的方案，作为评估肝脂肪变性动物模型中肝脏标本中 LD 数、面积和直径的替代分析方法。该程序包括肝脏样本制备、组织切片、染色条件、图像采集和数据分析。

<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

脂滴（LDs）是介导脂质储存的特化细胞器，在抑制脂毒性和预防游离脂肪酸（FAs）引起的功能障碍方面起着非常重要的作用。鉴于肝脏在人体脂肪代谢中的关键作用，肝脏一直受到微泡和大泡肝脂肪变性形式的LDs细胞内积累的威胁。LD的组织学表征通常基于脂溶性重氮染料，例如油红O（ORO）染色，但许多缺点一直阻碍了该分析在肝脏标本中的使用。最近，亲脂性荧光团 493/503 因其快速吸收和积累到中性脂滴核心中而变得流行用于可视化和定位 LD。尽管大多数应用在细胞培养中都有很好的描述，但很少有证据表明亲脂性荧光团探针在组织样品中可靠地用作LD成像工具。在本文中，我们提出了一种优化的基于硼二吡咯甲烷（BODIPY）493/503的方案，用于评估来自高脂肪饮食（HFD）诱导的肝脂肪变性动物模型的肝脏标本中的LD。该协议涵盖肝脏样本制备、组织切片、BODIPY 493/503 染色、图像采集和数据分析。我们证明了HFD喂养时肝LD的数量，强度，面积比和直径增加。使用正交投影和3D重建，可以观察到LD核心中性脂质的全部含量，这些脂质显示为近球形液滴。此外，使用荧光团BODIPY 493/503，我们能够区分微囊泡（1μm<d≤3μm），中间囊泡（3μm<d≤9μm）和大囊泡（d>9μm），从而成功区分微泡和大泡脂肪变性。总体而言，该基于荧光的BODIPY 493/503方案是肝LD表征的可靠且简单的工具，可能是经典组织学方案的补充方法。

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.

该技术的成功实施应导致清晰的脂滴染色，以同时表征LD形态（基于3D重建的形状和脂质核心密度）以及它们的空间分布，每总面积的数量和平均大小（使用上述管道进行评估， 图1）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65206/65206fig01.jpg"> 图 1：使用细胞分析器处理示例 图像?...

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

在本文中，我们展示了一种优化的基于BODIPY 493 / 503荧光的方案，用于肝组织中的脂滴表征。通过使用正交投影和3D重建，荧光团可以成功区分微泡和大泡脂肪变性，并且可能代表用于肝脂肪变性评估的经典组织学方案的补充方法。

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

这种成像方法允许肝脂滴沉积的三维重建，并克服了经典组织学染色的主要缺点，这些缺点缩小了微观和大泡脂肪变性之间的成功区分。微泡性脂肪变性的评估是使用金标准组织学染色，由于天然脂滴的破坏和偶尔融合，两者和油红。pro-BODIPY为微泡肝脂肪变性评估的常规方案提供了一种替代方案，因为它具有正确的疏水性，可快速摄取和有效标记脂滴，不仅对环境极性不敏感，而且选择性极强。这种成像方法与免疫化学技术相结合，具有更广泛的应用，可以同时检测其他亚细胞抗原，这些抗原可能推进脂滴的生物发生和动力学研究，为微泡和大泡肝脂肪变性奠定基础。

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

Research

JoVE Journal

Biochemistry

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

3.0K 次观看.  University of Coimbra. 这种成像方法允许肝脂滴沉积的三维重建，并克服了经典组织学染色的主要缺点，这些缺点缩小了微观和大泡脂肪变性之间的成功区分。微泡性脂肪变性的评估是使用金标准组织学染色，由于天然脂滴的破坏和偶尔融合，两者和油红。pro-BODIPY为微泡肝脂肪变性评估的常规方案提供了一种替代方案，因为它具有正确的疏水性，可快速摄取和有效标记脂滴，不仅对环境极性不敏?...

视频: 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  

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.

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

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.

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

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