Droste Isabel, Schuitema Erik, Khan Sajjad, Heldens Stijn, van Werkhoven Ben, Lidke Keith A, Stallinga Sjoerd, Rieger Bernd
Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands.
ESSD Software Engineering, Rotterdam, The Netherlands.
bioRxiv. 2024 Dec 11:2024.12.11.627909. doi: 10.1101/2024.12.11.627909.
Image quality in single molecule localization microscopy (SMLM) depends largely on the accuracy and precision of the localizations. While under ideal imaging conditions the theoretically obtainable precision and accuracy are achieved, in practice this changes if (field dependent) aberrations are present. Currently there is no simple way to measure and incorporate these aberrations into the Point Spread Function (PSF) fitting, therefore the aberrations are often taken constant or neglected all together. Here we introduce a model-based approach to estimate the field-dependent aberration directly from single molecule data without a calibration step. This is made possible by using nodal aberration theory to incorporate the field-dependency of aberrations into our fully vectorial PSF model. This results in a limited set of aberration fit parameters that can be extracted from the raw frames without a bead calibration measurement, also in retrospect. The software implementation is computationally efficient, enabling fitting of a full 2D or 3D dataset within a few minutes. We demonstrate our method on 2D and 3D localization data of microtubuli and nuclear pore complexes over fields of view (FOV) of up to 180 μm and compare it with spline-based fitting and a deep learning based approach.
单分子定位显微镜（SMLM）中的图像质量在很大程度上取决于定位的准确性和精确性。虽然在理想的成像条件下可实现理论上可获得的精度和准确性，但在实际情况中，如果存在（与视场相关的）像差，情况就会有所变化。目前，尚无简单方法来测量这些像差并将其纳入点扩散函数（PSF）拟合中，因此像差通常被视为恒定或完全被忽略。在此，我们引入一种基于模型的方法，无需校准步骤即可直接从单分子数据估计与视场相关的像差。通过使用节点像差理论将像差的视场依赖性纳入我们的全矢量PSF模型，这得以实现。这导致了一组有限的像差拟合参数，这些参数可以从原始帧中提取，即使是事后也无需进行珠标校准测量。该软件实现具有计算效率，能够在几分钟内对完整的二维或三维数据集进行拟合。我们在高达180μm视场（FOV）的微管和核孔复合体的二维和三维定位数据上展示了我们的方法，并将其与基于样条的拟合和基于深度学习的方法进行比较。
