Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico, United States of America.
PLoS One. 2013 May 22;8(5):e64320. doi: 10.1371/journal.pone.0064320. Print 2013.
Many cellular signaling processes are initiated by dimerization or oligomerization of membrane proteins. However, since the spatial scale of these interactions is below the diffraction limit of the light microscope, the dynamics of these interactions have been difficult to study on living cells. We have developed a novel high-speed hyperspectral microscope (HSM) to perform single particle tracking of up to 8 spectrally distinct species of quantum dots (QDs) at 27 frames per second. The distinct emission spectra of the QDs allows localization with ∼10 nm precision even when the probes are clustered at spatial scales below the diffraction limit. The capabilities of the HSM are demonstrated here by application of multi-color single particle tracking to observe membrane protein behavior, including: 1) dynamic formation and dissociation of Epidermal Growth Factor Receptor dimers; 2) resolving antigen induced aggregation of the high affinity IgE receptor, FcεR1; 3) four color QD tracking while simultaneously visualizing GFP-actin; and 4) high-density tracking for fast diffusion mapping.
许多细胞信号转导过程都是由膜蛋白的二聚化或寡聚化启动的。然而，由于这些相互作用的空间尺度低于光显微镜的衍射极限，因此很难在活细胞上研究这些相互作用的动态。我们开发了一种新型高速高光谱显微镜（HSM），可在每秒 27 帧的速度下对多达 8 种光谱上不同的量子点（QD）进行单粒子跟踪。QD 的独特发射光谱允许在探针以低于衍射极限的空间尺度聚集时以约 10nm 的精度进行定位。通过将多色单粒子跟踪应用于观察膜蛋白行为来证明 HSM 的功能，包括：1）表皮生长因子受体二聚体的动态形成和解离；2）解析高亲和力 IgE 受体 FcεR1 上抗原诱导的聚集；3）同时可视化 GFP-肌动蛋白的四色 QD 跟踪；4）用于快速扩散映射的高密度跟踪。
