1] Department of Biophysical Chemistry, Institute for Physical and Theoretical Chemistry, University of Braunschweig, Braunschweig, Germany. [2].
1] Biomolecular Spectroscopy and Single-Molecule Detection Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. [2].
Nat Methods. 2014 May;11(5):579-84. doi: 10.1038/nmeth.2919. Epub 2014 Apr 6.
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.
当用旋转线偏振光激发时，不同取向的荧光染料会在不同时间发出周期性信号。我们表明，测量固定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（50nm）(2)图像纳米区域，可以提供亚衍射分辨率（通过偏振解调的超分辨率，SPoD）。由于对取向分子进行有效激发的偏振角范围相当宽且不具体，我们通过同时用第二束具有垂直于激发光束的偏振的去激发光束照射来缩小该范围（激发偏振角变窄，ExPAN）。这缩短了周期性发射闪光的时间，使分子或纳米区域之间的区分更加容易。我们的方法既不需要产生纳米级的干涉结构，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标准的宽场显微镜和双光子扫描显微镜，对神经元棘突的精细结构细节进行成像。
