Institute of Computational and Mathematical Engineering, Stanford, California 94305, USA.
Opt Lett. 2013 May 1;38(9):1521-3. doi: 10.1364/OL.38.001521.
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°-5° is achieved in proof-of-concept experiments for both azimuthal (φ) and polar (θ) 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.
这封信提出了一种测量荧光定向固定单分子偶极子取向的方法，该方法使用一种特殊设计的相位掩模，称为“方形光瞳”，共轭于传统宽场显微镜的后焦平面。该方法利用偶极子发射器远场发射模式的空间各向异性，并使这种各向异性在像平面上能够进行定量分析。与通过将模拟与离焦或过度放大的图像进行匹配来推断取向的旧的图像拟合技术相比，方形光瞳方法对较小的建模差异和光学像差更稳健。在无需离焦的情况下，对于方位角（φ）和极角（θ），该方法在概念验证实验中实现了 1°-5°的精度。由于相位掩模是在液晶空间光调制器上实现的，该调制器可以在不对样品或成像系统进行任何机械干扰的情况下停用，因此该技术可以很容易地集成到明场成像研究中。
