Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization

Abstract

3D Single molecule localization microscopy relies on fitting of the individual molecules with a point spread function (PSF) model. The reconstructed images often show local squeezing or expansion in z. A common cause are depth-induced aberrations in conjunction with an imperfect PSF model calibrated from beads on a coverslip, resulting in a mismatch between measured PSF and real PSF. Here, we developed a strategy for accurate zlocalization in which we use the imperfect PSF model for fitting, determine the fitting errors and correct for them in a post-processing step. We present an open-source software tool and a simple experimental calibration procedure that allow retrieving accurate z-positions in any PSF engineering approach or fitting modality, even at large imaging depths.

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

“…In addition to the pixel size, our cell lines can be used to verify the axial calibration in 3D SMLM. This is typically challenging, as aberrations or imperfect calibration of the PSF model can lead to systematic and depth-dependent localization errors, especially when using oil objectives 31 . Moreover, the refractive index difference between oil and the aqueous sample leads to an image compressed in z.…”

“…Furthermore, the distance between the rings was correlated to the distance between NPC and coverslip ( Supplementary Figure 5a), indicating aberrations. After correcting for these aberration-induced fitting errors with a method we recently developed 31 , the z-dependence was reduced ( Supplementary Figure 5b) and the average corrected distance is d = 49.8 ± 1.9 nm. Based on these results we could calibrate the RIMF to be 0.79.…”

Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy

“…Samples and data for the STORM modality were prepared and acquired according to Li et al 16 . In short, homozygous Nup107-SNAP U2-OS cell lines were fixed and labeled with Alexa Fluor 647benzylguanine and imaged on a custom-built setup that contains a cylindrical lens in the emission path for astigmatic 3D localization.…”

“…The data were fitted using an experimental PSF model calibrated using a z-stack of beads that were immobilized on the coverslip 15 . Subsequently, fitting errors induced by the refractive index mismatch were corrected based on a calibration of beads immobilized in a gel 16 .…”

“…We applied our algorithm to experimental SMLM data of NPCs in fixed U2OS cells (Figure 2 & Supplementary Movies 1-3). Cells expressing Nup107-SNAP labeled with Alexa Fluor 647benzylguanine were imaged with three different SMLM techniques, 3D astigmatic PAINT (Supplementary Figure 5), 3D astigmatic STORM 15,16 and 4Pi STORM 17,18 . Figure 2a, e and i show the results of fusing 306, 356, and 750 segmented particles for the three modalities, which had an average number of localizations per particle of 88, 70, and 58, respectively.…”