Summary

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

Published: June 08, 2020
doi:

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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 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。 在 SCD 中执行 1:50 的细胞稀释。…

Representative Results

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?…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

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Cite This Article
Adhikari, S., Banerjee, C., Moscatelli, J., Puchner, E. M. Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking. J. Vis. Exp. (160), e60950, doi:10.3791/60950 (2020).

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