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生物学

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Published: December 01, 2016
doi:
10.3791/54774
,
1Department of Biology,South University of Science and Technology of China, Shenzhen, 2Mechanobiology Institute, Singapore, 3Department of Biomedical Engineering,National University of Singapore

Summary

我们提出了一个协议，用于干涉式光活化的定位显微镜（iPALM），3维单分子定位超分辨率显微镜方法的应用，以便在贴壁哺乳动物细胞的肌动蛋白细胞骨架的成像。这种方法允许的纳米结构特征，否则将通过常规的衍射限制的光学显微镜仍未解决基于光的可视化。

Abstract

荧光显微镜使细胞内的特定生物分子的直接可视化。但是，对于以往的荧光显微镜，空间分辨率由衍射图像平面和> 500nm的沿着光轴内限制为〜200纳米。其结果是，荧光显微镜早已严重中的超微结构特征的细胞内观察的限制。最近的超分辨率显微镜方法发展克服了这一限制。特别是，光开关的荧光团的到来使基于与定位超分辨率显微镜，其提供接近分子长度尺度的分辨率。在这里，我们描述了基于单分子定位显微镜和多相干涉三维超高分辨率显微镜方法，称为干涉光敏定位显微镜（iPALM）的应用程序。这种方法提供了几乎各向同性分辨率为20nm顺序在所有三个维度。可视化丝状肌动蛋白骨架，其中包括iPALM仪器的样品制备和操作方案，如下所述。这些协议也容易适应，并启发对其他超微结构细胞研究。

Introduction

复杂的细胞结构的可视化一直是不可或缺的生物的见解和发现。虽然荧光显微镜可以形象细胞高分子特异性，其分辨能力被衍射限定在图像平面〜200纳米（X，Y，或横向尺寸）和> 500nm的沿着光轴（z或轴向尺寸） 1,2。因此，超微结构特征观察历来限于电子显微镜（EM）。幸运的是，最近的超分辨率显微镜的发展已经绕过这个限制，使得在10空间分辨率- 100纳米范围1-6。具体地，超分辨率方法的基础上的单分子的定位，由缩略词如PALM（光敏定位显微镜）4，FPALM（荧光光敏定位显微镜） 图5（d）风暴（直接随机光学重构显微术）6,7，涂料（已知宝成像纳米形貌）8 INT积累，GSDIM（基态耗尽显微术，随后由个别分子返程）9，或SMACM（单分子有源控制显微镜）10，以及它们的3维（3D）实现方式中，如干涉PALM（iPALM）11或3D风暴12日 ，在揭示新的见解无数的生物结构的纳米级组织，包括神经元轴突已经有价值和突触13，粘着斑14,15，细胞间的连接处16，核孔17和中心体18-20，仅举几例。

在这超分辨率显微镜是潜在有用的细胞超微结构的另一个特点是肌动蛋白骨架。丝状（F）肌动蛋白在细胞皮质复杂小梁在细胞形状和机械性能21的控制中起重要作用。该组织为OF F肌动蛋白积极并动态调节尽管这强烈地影响聚合，交联，周转率，稳定性，和网络拓扑22众多调节蛋白。然而，尽管F-肌动蛋白小梁结构的特征为机械见解重要成不同范围的细胞过程中，小尺寸（〜8纳米）的f微丝阻碍通过常规衍射限制光镜的观察;因此，肌动蛋白的精细结构的可视化迄今完全由EM执行。在这里，我们描述了可视化的贴壁哺乳动物细胞中的F-肌动蛋白细胞骨架，使用iPALM超分辨率显微镜技术，以利用其精度非常高的能力，在3D 11,23协议。虽然iPALM仪器高度专业化，对设立这样一台仪器指令已经描述了最近23，同时获得由何主办的iPALM显微镜病房休斯医学研究所也已提供给以最小的成本研究团体。此外，本文描述的样品制备方法是直接适用于替代三维超分辨率方法中，如根据点扩散函数（PSF）的象散散焦那些12或双平面检测24，这是更广泛地可用。

我们注意到，在一般的基于定位单分子超分辨率显微镜的必要成分是光开关荧光团25，其允许基于本地化单分子超分辨率显微镜三个重要的要求必须满足：ⅰ）高的单分子亮度和对比度相对于背景信号; II）在给定的图像帧单分子分布稀疏;和iii）高贴标足以捕获底层结构（也称为奈奎斯特沙的轮廓空间密度n非取样标准）26。因此，对于满意的结果，应强调同样在两个试样的适当准备放置优化荧光光控和维护底层超微结构，以及在实验的仪器和采集方面。

Protocol

1.成像试样制备因为背景的荧光信号与来自荧光团标签荧光干扰，通过在去离子水漂洗它们（DDH 2 O的），然后使用压缩空气空气干燥它们清洗盖玻片。接着，在等离子体清洁器执行等离子体蚀刻15秒，或更长的时间，如果必要的。 为使漂移校正和iPALM校准，使用＃1.5圆（22毫米直径）嵌入荧光纳米粒子作为基准标记，供应可靠校准和漂移校正的高度光基准预清洁盖玻片。由?…

Representative Results

对于iPALM关键要求是对准，配准，并且光学系统的校准。这些是必要的，以确保3路分束器位于必要的适当的干扰为z坐标萃取。要启用连续监测，荧光不断的点源是必要的。这可以通过使用荧光Au或双金属纳米粒子23的光致发光从局域型表面等离子体共振（LSPR）出现而实现。它们作为在照明用的稳定的单偶极子和通常可以用5本地化 – 10纳米的精度。这些可商购的纳?…

Discussion

iPALM的光学系统是基于一个4π双相对目标设计， 如图1A所示 。设置同时使用定制加工和商业光学机械部件构成，如表 1中所述前面23和列出。除了我们的设置中，霍华德休斯医学研究所（HHMI）举办一个系统，是在高级影像中心，科学界在珍利亚农场研究园区访问。对于整个机械图纸，控制原理图和软件，我们鼓励读者在HHMI与哈拉尔赫斯询问进一步的?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

YW和PK非常感谢来自新加坡国家研究基金会，授予PK（NRF-NRFF-2011-04和NRF2012NRF-CRP001-084）的资金支持。我们也感谢对基础设施的支持MBI开放实验室，显微镜的核心设施。

Materials

optical table Newport, CA  RS4000 iPALM, installed on 4 Newport Stabilizer vibration isolators 
vibration isolator for optical table Newport, CA  S-2000 
laser-642 Newport, CA  1185055 output power=100mw
laser-561 Newport, CA  1168931 output power=200mw
laser-488 Newport, CA  1137970 output power=200mw
laser-405 Newport, CA  1142279 output power=100mw
broadband dielectric mirrors  Thorlabs, NJ BB1-E02 laser combiner
dichroic beamsplitter  Semrock, NY LM01-427-25
acousto-optic tunable filter  AA Opto-Electronic, France AOTFnC-VIS-TN
Linear polarizer Newport, CA  05LP-VIS-B
baseplate local workshop customized
turning mirror (22.5°) Reynard Corpporation, CA customized 22.5° mirror
motorized optic mounts  New Focus, CA 8816
motorized XYZ translation stage Thorlabs, NJ MT3/M-Z6 sample holder
T-Cube DC servo motor controller Thorlabs, NJ TDC001
Piezo Phase Shifter Physik Instrumente, Germany S-303.CD
objective lens Nikon, Japan MRD01691 objective. Apo TIRF 60X/1.49oil
translation stage New Focus, CA 9062-COM-M
Pico Motor Actuator New Focus, CA 8301
rotary Solenoid/Shutter DACO Instruments, CT 5423-458
3-way beam splitter Rocky Mountain Instruments, CO customized beamsplitter
Piezo Z/Tip/Tilt scanner Physik Instrumente, Germany S-316.10
motorized five-axis tilt aligner  New Focus, CA 8081
Picmotor ethernet controller New Focus, CA 8752
Piezo controllers/amplifier/digital operation module Physik Instrumente, Germany E-509/E-503/E-517
band-pass filter Semrock, NY FF01-523/20 filters
band-pass filter Semrock, NY FF01-588/21
band-pass filter Semrock, NY FF01-607/30
band-pass filter Semrock, NY FF01-676/37
notch filter Semrock, NY NF01-405/488/561/635
motorized filter wheel with controllter Thorlabs, NJ FW103H
EMCCD Andor, UK  DU-897U-CSO-#BV 3 sets
Desktop computers for controlling cameras and synchronization Dell Precision T3500 PC, 4 sets
coverslips with fiducial Hestzig, VA 600-100AuF sample preparation. fiducial marks with various density and spectra  available
fibronectin  Millipore, MT FC010
paraformaldehyde Electron Microscopy Sciences, PA 15710 fixation. 16%
glutaraldehyde  Electron Microscopy Sciences, PA 16220 25%
triton X-100 Sigma aldrich, MO T8787
HUVEC cells Life Technologies, CA C-015-10C
Medium 200 Life Technologies, CA M-200-500
Large Vessel Endothelial Factors Life Technologies, CA A14608-01
Dulbecco's Phosphate Buffered Saline 14190367
Pennicillin/Streptomycin 15140122
Trypsin/EDTA Life Technologies, CA 25200056
PIPES Sigma aldrich, MO P1851 PHEM
HEPES 1st base, Malaysia BIO-1825
EGTA Sigma aldrich, MO E3889
MgCl2 Millipore, MT 5985
 Alexa Fluor 647 Phalloidin Invitrogen, CA A22287 staining
sodium borohydride (NaBH4)  Sigma aldrich, MO 480886 quenching
glucose 1st base, Malaysia BIO-1101 imaging buffer
glucose oxidase Sigma aldrich, MO G2133
catalase Sigma aldrich, MO C9322
cysteamine  Sigma aldrich, MO 30070
Epoxy Thorlabs, NJ G14250
vaseline Sigma aldrich, MO 16415 sample sealing
lanolin Sigma aldrich, MO L7387
parafin wax Sigma aldrich, MO 327204
Immersion oil  Electron Microscopy Sciences, PA 16915-04 imaging. Cargille Type HF
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Tags

Keywords: 3D Super Resolution MicroscopyIPALMF-actin FilamentsUltrastructural VisualizationSingle Molecule LocalizationCell Sample PreparationImaging BufferCover Glass AssemblyEpoxy SealingNewton’s Rings Pattern
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Wang, Y., Kanchanawong, P. Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM). J. Vis. Exp. (118), e54774, doi:10.3791/54774 (2016).
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