Accurate 3D single-molecule localization via vectorial <i>in situ</i> point spread function retrieval and aberration assessment

Single-molecule localization microscopy (SMLM) gradually plays an important role in deep tissue imaging. However, current SMLM methods primarily rely on fiducial marks, neglecting aberrations introduced by thick samples, thereby resulting in decreased image quality in deep tissues. Here, we introduce vectorial in situ point spread function (PSF) retrieval (VISPR), a method that retrieves a precise PSF model considering both system- and sample-induced aberrations under SMLM conditions. By employing the theory of vectorial PSF model and maximum likelihood estimation (MLE) phase retrieval, VISPR is capable of reconstructing an accurate in situ 3D PSF model achieving the theoretically minimum uncertainty and accurately reflecting three-dimensional information of single molecules. This capability enables accurate 3D super-resolution reconstruction in deep regions away from the coverslips. Additionally, VISPR demonstrates applicability in low signal-to-noise ratio scenarios and compatibility with various SMLM microscope modalities. From both simulations and experiments, we verified the superiority and effectiveness of VISPR. We anticipate that VISPR will become a pivotal tool for advancing deep tissue SMLM imaging.