Single-molecule orientation measurements with a quadrated pupil
Single-molecule orientation measurements with a quadrated pupil
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DOI：
10.1364/ol.38.001521
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发表时间：
2013-05-01
期刊：
影响因子：
3.6
通讯作者：
Moerner, W. E.
中科院分区：
文献类型：
--
作者：
Backer, Adam S.;Backlund, Mikael P.;Moerner, W. E.
这封信提出了一种测量荧光、定向固定单分子偶极子方向的方法，该方法使用专门设计的相位掩模，称为“四方光瞳”，与传统宽视场显微镜的后焦平面共轭。该方法利用偶极子发射器远场发射图案的空间各向异性，并使这种各向异性适合在图像平面上进行定量分析。与通过将模拟与散焦或过度放大的图像相匹配来推断方向的旧图像拟合技术相比，方形光瞳方法对于微小的建模差异和光学像差更加稳健。在方位角 (phi) 和极角 (theta) 的概念验证实验中，在不散焦的情况下实现了 1 度至 5 度的精度。由于相位掩模是在液晶空间光调制器上实现的，可以在不对样品或成像系统进行任何机械扰动的情况下停用该调制器，因此该技术可以很容易地集成到通光孔径成像研究中。 (C) 2013 美国光学学会
This Letter presents a means of measuring the dipole orientation of a fluorescent, orientationally fixed single molecule, which uses a specially designed phase mask, termed a "quadrated pupil," conjugate to the back focal plane of a conventional wide-field microscope. The method leverages the spatial anisotropy of the far-field emission pattern of a dipole emitter and makes this anisotropy amenable to quantitative analysis at the image plane. In comparison to older image-fitting techniques that infer orientation by matching simulations to defocused or excessively magnified images, the quadrated pupil approach is more robust to minor modeling discrepancies and optical aberrations. Precision of 1 degrees-5 degrees is achieved in proof-of-concept experiments for both azimuthal (phi) and polar (theta) angles without defocusing. Since the phase mask is implemented on a liquid-crystal spatial light modulator that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into clear aperture imaging studies. (C) 2013 Optical Society of America