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Single-Molecule Fluorescence Imaging on the Cell Membrane


Single-Molecule Fluorescence Imaging on the Cell Membrane Using a Super High Numerical Aperture (NA) Objective Lens

Introduction

Recent advances in cell preparation and microscope optical systems have enabled imaging of single biomolecules in a live cell. Molecular dynamics, such as the binding of a physiologically active ligand to a cell, dimerization of signal molecules, and the formation of a molecular complex, can be visualized at the single molecule level in live a cell using objective lenses with a super high numerical aperture. In this study, researchers used an Olympus super high NA objective lens for fluorescence imaging of intermolecular interactions in ion channels on the cell membrane at the single molecule level.

Super high NA objective lens TIRF application

Fluorescent-protein (FP) tagged ion channel subunits are expressed in Xenopus oocytes and observed at the single molecule level by TIRF microscopy (Figure 1, left). Stochastic bleaching events of individual FPs can be observed as ‘bleaching steps’ (Figure 1, right). The number of subunits in a single ion channel complex can be determined by counting the bleaching steps from individual fluorescent spots.

the subunit counting
Figure 1. A schematic overview of the subunit counting by single molecule photobleaching. The photobleaching steps are represented by the green arrows.

 the single molecule level under TIRF microscopy
Figure 2. Images of the fluorescent proteins Kv4.2-mCherry (left) and mEGFP-DPP10 (middle) expressed in Xenopus oocyte at the single molecule level under TIRF microscopy. 

Images of the fluorescent proteins Kv4.2-mCherry Figure 2, left) and mEGFP-DPP10 (Figure 2, middle) expressed in a Xenopus oocyte were observed at the single molecule level using TIRF microscopy and super high NA objective lenses.  Each red spot represents a single Kv4.2 channel (tetramer).  Some of the green spots overlap with the red spots (white arrowheads in Figure 2, right) indicating that Kv4.2 and DPP10 form a complex.  By counting bleaching events of mEGFP from a single fluorescent spot, the number of subunits in the complex can be counted. 1  In the Figure 2 graph, four bleaching events (green arrows) were observed from a Kv4.2-mCherry/mEGFP-DPP10 spot, suggesting four DPP10 subunits were included in the complex.
1 Ulbrich, Maximilian H., and Ehud Y. Isacoff. “Subunit counting in membrane-bound proteins.” Nature methods 4, no. 4 (2007): 319–321

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Movie of Kv4.2-mCherry and mEGFP-DPP10

 


Movie of Kv4.2-mCherry and mEGFP-DPP10

Imaging System;
Microscope: Research Inverted Microscope IX71
Objective:  Apo 100XOHR (100X, N.A.1.65)
Ex: 488nm (Solid laser, Spectra-Physics) , 588nm (Solid laser, Coherent)
CCD camera: iXon3 EMCCD camera (Andor)
Coverslips: High refractive index coverslip (n = 1.78)

Image data courtesy of;
Masahiro Kitazawa, Ph.D., Yoshihiro Kubo, M.D.,Ph.D., Koichi Nakajo*, Ph.D.
Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences
*Present address:  Department of Physiology, Osaka Medical College

Reference;
J Biol Chem. 2015 Sep 11; 290(37):22724-33. doi: 10.1074/jbc.M115.646794.
J Biol Chem. 2014 Jun 20;289(25):17597-609. doi: 10.1074/jbc.M114.563452.
Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18862-7. doi: 10.1073/pnas.1010354107.

Conclusion

A high numerical aperture objective lens designed only for evanescent illumination can produce remarkably high contrast images even with weak fluorescent light because of the efficient formation of an evanescent wave field with a shallow penetration depth. While this type of observation requires the quantitative measurement of minute changes in fluorescence intensity due to fluorescence loss at the single molecule level, observation using a super high NA objective lens with special immersion oil and coverslips facilitates images of intermolecular interactions in ion channels in Xenopus oocyte membranes. Since these images have a high signal-to-noise ratio, changes in fluorescence intensity can be quantitatively measured.

Products used for this application

出色的多色 TIRF 成像

IXplore TIRF

  • 出色的同步多色 TIRF,可用于调查膜动力学和单分子检测
  • 由于具备穿透深度控制,可以实现最多四个标记的精确共定位
  • 利用奥林巴斯卓越的 TIRF 物镜,可实现全球最高 NA,高达 1.7*
  • 利用图形化试验管理系统 (GEM) 可以直观设置复杂的试验
用于超高分辨率成像或全内反射成像的高分辨率物镜

APON-TIRF/UAPON-TIRF/UPLAPO-HR

  • 高数值孔径为高对比度要求的全内反射成像或超高分辨率成像提供极佳的消失波
  • HR系列物镜是世界首款*高平场性下数值孔径达到1.5的平场复消色差物镜。

* 根据奥林巴斯的研究,截止至2018年11月

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