Interferometric single-molecule localization microscopy (iPALM, 4Pi-SMS) uses multiphase interferometry to localize single fluorophores and achieves nanometer isotropic resolution in 3D. The current data analysis workflow, however, fails to reach the theoretical resolution limit due to the suboptimal localization algorithm. Here, we develop a method to fit an experimentally derived point spread function (PSF) model to the interference 4Pi-PSF. As the interference phase is not fixed with respect to the shape of the PSF, we decoupled the phase term in the model from the 3D position of the PSF. The fitter can reliably infer the interference period even without introducing astigmatism, reducing the complexity of the microscope. Using a spline-interpolated experimental PSF model and by fitting all phase images globally, we show on simulated data that we can achieve the theoretical limit of 3D resolution, the Cramér–Rao lower bound (CRLB), also for the 4Pi microscope.

中文翻译：

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使用实验性 PSF 模型准确定位 4Pi 单分子。

干涉单分子定位显微镜（iPALM、4Pi-SMS）使用多相干涉测量法来定位单个荧光团，并在 3D 中实现纳米各向同性分辨率。然而，由于定位算法不理想，当前的数据分析工作流程未能达到理论分辨率极限。在这里，我们开发了一种将实验导出的点扩散函数 (PSF) 模型拟合到干扰 4Pi-PSF 的方法。由于干扰相位相对于 PSF 的形状不固定，我们将模型中的相位项与 PSF 的 3D 位置分离。即使不引入像散，安装人员也可以可靠地推断出干涉周期，从而降低显微镜的复杂性。使用样条插值实验 PSF 模型并通过全局拟合所有相位图像，