Name: conventional bodipy conjugates for live-cell super-resolution microscopy and single-molecule tracking
Uploaded: 2020-06-08T13:00:00
Description: Watch this Scientific Journal Video about Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking at JoVE.com

本出版物中报告的研究得到了国家卫生研究院国家普通医学研究所的支持，编号为R21GM127965。

注：关于酵母克隆和内源性标记，请参阅我们最近的出版物10。
1. 制备用于成像的酵母细胞样品
<ol><li>准备w303酵母菌株的液体过夜培养。使用无菌木棍，从含有酵母提取物的a加盘（peptone_dextrose）中发现少量酵母细胞，放入含有±2 mL合成完整脱脂（SCD）介质的培养管中。在 270 rpm 和 30°C 的摇摇培养箱中孵育管过夜。</li>
	<li>在 SCD 中执行 1：50 的细胞稀释。...

<ol>
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在此协议中，我们演示了如何使用传统的 BODIPY 偶联来获取具有空间分辨率级级改进的 SMLM 图像。该方法基于利用先前报告的红色移位DII状态的传统BODIPY染料，这些状态通过双分子接触暂时形成。这些状态可以特别兴奋和检测与红色偏移波长，是稀疏和短寿命足以SMLM。通过调整 BODIPY 单体与激光参数的浓度，可实现本地化和噪声到噪声的最佳密度。在喂养和禁食条件下，我们解决了脂肪酸...

单分子定位显微镜（SMLM）技术，如随机光学重建显微镜（STORM）和光活化定位显微镜（PALM），已成为生成超分辨率图像的方法，其信息超过Abbe的光衍射极限11，2，2并跟踪单生物分子33，44的动态。与 SMLM 兼容的探头要求之一是能够随时控制活动荧光波的数量，以避免其点差函数 （PSF） 的空间重叠。在数千个数据采集帧中，通过安装其相应的点扩散功能，确定每个荧光荧光波的定位，精度为±20 nm。传统上，氟脑的开离闪烁是通过随机光切换11，2，52,5或化学诱导的内在闪烁6加以控制。其他方法包括荧光原在瞬态结合时诱导激活氟原蛋白77、8，8以及标记的DNA寡聚物在总内部反射荧光（TIRF）或光片激发9中的可编程结合。最近，我们报道了SMLM10的新颖和通用的标签策略，其中先前报道的红移二恶热（DII）状态的传统硼二丙甲甲（BODIPY）结合11，12，13是瞬时形成，并成为特别兴奋和检测与红移波长。11,12,13
BODIPYs是广泛使用的染料与数百种变种，专门标记亚细胞隔间和生物分子14，15，16。14,15,16由于其易用性和适用于活细胞，BODIPY 变体在商业上可用于传统荧光显微镜。在这里，我们描述了一个详细和优化的协议，如何将数以百计的商用 BODIPY 联结物用于活细胞 SMLM。通过调整BODIPY单体浓度，优化激发激光功率、成像和数据分析参数，在活细胞中获得高质量的超分辨率图像和单分子跟踪数据。当在低浓度（25-100 nM）下使用时，BODIPY偶联剂可同时用于红移通道中的SMLM和传统发射通道中的相关传统荧光显微镜。可以分析获得的单分子数据，以量化不动结构的空间组织，提取活细胞17中分子的扩散状态。具有绿色和红色两种形式的 BODIPY 探头，可在与其他兼容荧光波的正确组合中使用多色成像。
在本报告中，我们提供了一个优化的协议，用于使用BODIPY-C 12、BODIPY（493/503）、BODIPY-C12红色和多种颜色的色解解解器绿色来获取和分析活细胞SMLM数据。12我们以±30nm的分辨率解决活酵母和哺乳动物细胞中的脂肪酸和中性脂质。我们进一步证明，酵母细胞根据它们的代谢状态调节外部添加脂肪酸的空间分布。我们发现，在喂食条件下，在内质视网膜（ER）和脂液（LD）中加入的BODIPY-脂肪酸（FA）局部化，而BODIPY-FA在禁食后在血浆膜中形成非LD簇。进一步扩展了该技术在活体哺乳动物细胞中图像的脂体和LD的应用。我们使用传统的 BODIPY 联偶联体优化的 SMLM 协议是利用无数可用的 BODIPY 偶联物在纳米尺度上研究生物过程的有用资源。

<table border="0" cellpadding="0" cellspacing="0" style="width:1009px;" width="1008">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BODIPY C12</td>
			<td style="width:196px;">ThermoFisher</td>
			<td style="width:173px;">D3822</td>
			<td style="width:265px;">Green fatty acid analog</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BODIPY C12 Red</td>
			<td style="width:196px;">ThermoFisher</td>
			<td style="width:173px;">D3835</td>
			<td>Red fatty acid analog</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BODIPY(493/503)</td>
			<td style="width:196px;">ThermoFisher</td>
			<td style="width:173px;">D3922</td>
			<td>Neutral lipid marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concanavalin A</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:173px;">C2010</td>
			<td>Cell immobilization on glass surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Drop-out Mix Complete w/o nitrogen base</td>
			<td style="width:196px;">US Biological</td>
			<td style="width:173px;">D9515</td>
			<td>Amino acids for SCD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Dextrose</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:173px;">G7021</td>
			<td>Carbon source for SCD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Eight Well</td>
			<td style="width:196px;">Cellvis</td>
			<td style="width:173px;">C8-1.58-N</td>
			<td>Chambered Coverglasses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Eight Well, Lb-Tek II</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:173px;"></td>
			<td>Chambered Coverglasses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ET525/50</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Bandpass filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ET595/50</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Bandpass filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ET610/75</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Bandpass filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:173px;">26140079</td>
			<td>Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">FF652</td>
			<td style="width:196px;">Semrock</td>
			<td style="width:173px;"></td>
			<td>Beam splitter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">FF731/137</td>
			<td style="width:196px;">Semrock</td>
			<td style="width:173px;"></td>
			<td>Bandpass filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluoroBrite DMEM</td>
			<td style="width:196px;">ThermoFisher</td>
			<td>A1896701</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Hal4000</td>
			<td style="width:196px;">Zhuang Lab, Harvard University</td>
			<td style="width:173px;"></td>
			<td>Data acquisition software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ixon89Ultra DU-897U</td>
			<td style="width:196px;">Andor</td>
			<td style="width:173px;"></td>
			<td>EMCCD camera for photon detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Laser 405, 488, 561, 640 nm</td>
			<td style="width:196px;">CW-OBIS</td>
			<td style="width:173px;"></td>
			<td>Lasers for excitation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Insight3</td>
			<td style="width:196px;">Zhuang Lab, Harvard University</td>
			<td style="width:173px;"></td>
			<td>Single molecule localization software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">L-Glutamine</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:173px;">25030-081</td>
			<td>Amino acid required for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">live-cell imaging solution</td>
			<td style="width:196px;">ThermoFisher</td>
			<td>A14291DJ</td>
			<td>Imaging buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Lysotracker Green</td>
			<td style="width:196px;">ThermoFisher</td>
			<td style="width:173px;">L7526</td>
			<td>Bodipy based lysosome marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mammalian ATCC U2OS cells (Manassas, VA)</td>
			<td colspan="2">Dr. Jochen Mueller (University of Minnesota)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon-CFI Apo 100 1.49 N.A</td>
			<td>Nikon</td>
			<td style="width:173px;"></td>
			<td>Oil immersion objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin</td>
			<td style="width:196px;">Gibco</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium Pyruvate</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:173px;">11360-070</td>
			<td>Supplement for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">T562lpxr</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Beam splitter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA</td>
			<td style="width:196px;">Gibco</td>
			<td>15400-054</td>
			<td>Dissociation of adherent cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">W303 MATa strain</td>
			<td style="width:196px;">Horizon-Dharmacon</td>
			<td style="width:173px;">YSC1058</td>
			<td>Parental yeast strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Yeast Nitrogen Base</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:173px;">Y1250</td>
			<td>Nitrogen base without amino-acids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">zt405/488/561/640rdc</td>
			<td style="width:196px;">Chroma</td>
			<td style="width:173px;"></td>
			<td>Quadband dichroic mirror</td>
		</tr>
	</tbody>
</table>

conventional bodipy conjugates for live-cell super-resolution microscopy and single-molecule tracking

单分子定位显微镜（SMLM）技术克服了传统荧光显微镜的光衍射极限，能够以±20nm的精度解决细胞内结构和生物分子的动态。SMLM 的先决条件是荧光器，它从黑暗状态过渡到荧光状态，以避免其点扩散函数在数千个数据采集帧中每个帧中的分时重叠。BODIPYs 是成熟的染料，在传统显微镜中使用大量结合物。红移BODIPY地面状态变暗器（DII）的瞬态形成导致明亮的单分子发射，使单分子局部显微镜（SMLM）成为可能。在这里，我们提出了一个简单但通用的SMLM协议与传统的BODIPY结合在活酵母和哺乳动物细胞。此过程可用于获取超分辨率图像并跟踪单个 BODIPY-DII状态，以提取 BODIPY 偶联的时空信息。我们应用这个程序来解决脂质滴（LDs）、脂肪酸和在纳米长度尺度上活的酵母和哺乳动物细胞中的脂质脂滴（LD）、脂肪酸和淋索体。此外，我们演示了 BODIPY 染料与其他荧光探头结合使用时的多色成像功能。我们的代表性结果表明，在喂养和禁食条件下，酵母中BODIPY脂肪酸和中性脂质的空间分布和流动性存在差异。这种针对 SMLM 的优化协议可用于数百种商用 BODIPY 偶联，是研究纳米尺度生物过程的有用资源，远远超出本工作的应用范围。

在这里，我们提出了基于上述协议（图1A）的基于BODIPY偶联的SMLM优化的样品制备、数据采集和分析程序。为了演示获取和分析 SMLM 数据的工作流示例，我们在酵母中使用 BODIPY （493/503） 来解决光学衍射限制以下的 LD（图 1B-F）。图2显示了BODIPY与其他探测器（如GFP、mEos2）不同的多色成像模式的示例。我们操纵酵母的?...

Watch this Scientific Journal Video about Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking at JoVE.com

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

传统的BODIPY结合物可用于活细胞单分子定位显微镜（SMLM），其瞬时成型、红移地面状态变暗器。我们提出了优化的SMLM协议，以跟踪和解决活哺乳动物和酵母细胞中的亚细胞中性脂质和脂肪酸在纳米长度尺度上。

用于活细胞超分辨率显微镜和单分子跟踪的传统 BODIPY 结合物

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

我们的协议描述了使用传统的BODIPY染料使用其稀疏的红移状态进行超分辨率显微镜。这使我们能够研究活细胞中具有30纳米分辨率的细胞和生物分子。这种方法的优点在于其简单性和不同可用的BODIPY结合的多功能性，提供了单分子信号的持久源。通过解决脂质液滴和脂肪酸低于衍射极限，我们证明的应用对于在脂肪肝疾病或2型糖尿病等疾病中获得见解变得非常重要。我们在酵母和哺乳动物细胞的脂肪酸和脂滴生物学方面获得了深入的见解。然而，该技术适用于所有其他具有低背景荧光的透明细胞类型。首次使用技术时，请确保优化二浓度和激光功率，以观察明亮的单分子信号。这种技术的视觉演示对于了解激光功率的二浓度和优化如何产生明亮的单分子信号非常重要。在非荧光DMBM中保持哺乳动物U2OS细胞，用10%的胎儿牛血清4毫摩尔谷氨酰胺、1毫摩尔丙酮钠和1%青霉素链霉素抗生素在T-25烧瓶中。将细胞在70至80%的汇合下分裂为1至5，并在8个井板的单井中移液器。改变八井板中的细胞12至24小时。成像前十分钟，添加一个BODIPY-C12 Lysotracker绿色或其他BODIPY结合物，添加100纳米摩尔的最终浓度。根据使用的 BODIPY 的发射颜色在发射路径中安装相应的滤波器集。打开显微镜。显微镜级、488 纳米和 561 纳米激光器以及相机。在显微镜目标上添加一滴新兴油。打开 HAL4000 软件，该软件可控制 LED 灯，用于用于明亮场成像、激光电源、激光百叶窗和相机设置的成像功能。将 EMCCD 增益设置为 30，将摄像机温度设置为零下 68 摄氏度。准备相机和相应的软件，以20赫兹录制电影。打开显微镜舞台加热器，将其设置为 37 摄氏度的温度和 5% 的二氧化碳水平。相应地调整目标校正颜色。将样品安装在显微镜阶段并对焦，直到对焦系统接合。使用舞台控制器移动舞台，直到视图字段中出现健康的单元格。加载激光快门序列，用于激发一元和调光器，其中561纳米激光的激光功率为每平方厘米821千瓦，用于单个分子定位显微镜，因此，在红移发射通道中检测到单分子荧光孔。调整488纳米激光每平方厘米点035和点07瓦之间的激光功率，使常规荧光出现在绿色发射通道中。选择电影的目标文件夹并录制 5000 到 20000 个采集帧，以收集足够的本地化以重建超分辨率图像。移动到不同的视图区域并重复以收集更多单元格的数据。将影片加载到单个分子定位显微镜分析软件中。目视电影并调整对比度设置，以便可见单分子荧光闪烁。设置单分子识别参数，用于拟合 2D 高斯、PSF。目视筛选通过一些示例帧来检查识别参数，并可靠地检测出不同的单分子荧光爆发。按压分析，执行单分子定位显微镜想象分析与优化的识别参数。然后，将每个单个分子渲染为 2D 高斯分子，其宽度由检测到的光子数的反向平方根加权。评估数据的质量。使用受限帧范围观察单个分子在更具体实例和时间分布。这就占了数据采集过程中细胞器运动的影响。在这项研究中，我们提出了一个优化的样品制备，数据采集和分析程序与单分子定位显微镜使用BODIPY结合。为了演示获取和分析单分子定位显微镜数据的工作流程示例，利用酵母中的BODIPY来解析低于光学衍射极限的脂质液滴。此处显示了BODIPY与其他探头（如GGFP和mEos2）结合的不同多色成像模式的示例。BODIPY-C12在细胞外围的移动非脂质液滴群中形成，与在进给条件下将其并入脂液滴形成鲜明对比。为了进一步将BODIPY结合的单分子定位显微镜功能扩展到哺乳动物细胞，对活U2OS细胞中的BODIPY-C12和Lysotracker绿色进行了成像。优化BODIPY浓度以及刺激激光功率是可视化明亮的单分子信号和重建超分辨率图像的关键步骤。该方法可与任何其他BODIPY结合物一起使用，在活细胞内特定生物分子的特殊时间分布中获得高分辨率洞察力。我们的技术铺平了道路，进一步询问脂质液滴和脂肪酸生物学的纳米透镜规模。然而，由于各种功能BODIPY结合的可用性，此应用程序远远超出了特定的哲学。请务必遵循处理生物样品和染料的标准操作程序，同时遵守激光安全程序。

Research

JoVE Journal

Biochemistry

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

在质膜用新型荧光探针磷脂酰-4,5-二磷酸的单分子超分辨率成像

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

科研

生物化学

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

磁性镊子 quadruplexes 单分子操作

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

使用开源 smfBox 进行精确和准确的单分子 FRET 测量

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...