Fluorescence nanoscopy by polarization modulation and polarization angle narrowing
Fluorescence nanoscopy by polarization modulation and polarization angle narrowing
复制标题
DOI：
10.1038/nmeth.2919
复制
发表时间：
2014-05-01
期刊：
影响因子：
48
通讯作者：
Walla, Peter J.
中科院分区：
文献类型：
--
作者：
Hafi, Nour;Grunwald, Matthias;Walla, Peter J.
在旋转的线偏振光激发下，不同取向的荧光染料发出的周期信号在不同的时间达到峰值。我们表明，测量附着在刚性样品结构上的荧光染料的平均取向，映射到规则定义的（50 nm）(2)图像纳米区域，可以提供亚衍射分辨率（偏振解调的超分辨率，SPoD）。由于定向分子有效激发的偏振角范围相当宽且不特定，我们通过与具有垂直于激发束的偏振的第二束去激发光束同时辐照来缩小这个范围（激发偏振角狭窄，ExPAN）。这缩短了周期性发射闪光，使分子或纳米区域之间的区分更好。我们的方法既不需要产生纳米干涉结构，也不需要使用可切换或闪烁的荧光探针。我们将该方法应用于带相机检测的标准宽视场显微镜和双光子扫描显微镜，对神经元棘的精细结构细节进行成像。
When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)(2) image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, SPoD). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, ExPAN). This shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. Our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.