Reconstruction From Multiple Particles for 3D Isotropic Resolution in Fluorescence Microscopy

Abstract

The imaging of proteins within macromolecular complexes has been limited by the low axial resolution of optical microscopes. To overcome this problem, we propose a novel computational reconstruction method that yields isotropic resolution in fluorescence imaging. The guiding principle is to reconstruct a single volume from the observations of multiple rotated particles. Our new operational framework detects particles, estimates their orientation, and reconstructs the final volume. The main challenge comes from the absence of initial template and a priori knowledge about the orientations. We formulate the estimation as a blind inverse problem, and propose a block-coordinate stochastic approach to solve the associated non-convex optimization problem. The reconstruction is performed jointly in multiple channels. We demonstrate that our method is able to reconstruct volumes with 3D isotropic resolution on simulated data. We also perform isotropic reconstructions from real experimental data of doubly labeled purified human centrioles. Our approach revealed the precise localization of the centriolar protein Cep63 around the centriole microtubule barrel. Overall, our method offers new perspectives for applications in biology that require the isotropic mapping of proteins within macromolecular assemblies.

Homogeneous multifocal excitation for high-throughput super-resolution imaging

“…The high throughput offered by mfFIFI-iSIM allowed us to collect 3D images of thousands of purified expanded Chlamydomonas reinhardtii centrioles per hour ( Supplemental Figure 8, Supplemental Table 2 ). Having obtained 3D rather than 2D particles allowed us to use fewer particles to perform averaging and high-resolution multicolor reconstruction 44 compared to previous approaches using STORM 45 or electron microscopy 46,47 . Thus, we classified particles before reconstruction to identify and average only those particles sharing a high degree of similarity (ranging from 10-50% of the whole dataset), and thereby achieve even higher resolution reconstructions ( Figure 4f-h, Supplemental information, Supplemental Figure 9 ).…”

Imaging beyond the super-resolution limits using ultrastructure expansion microscopy (UltraExM)

“…To gain more insight into PolyE distribution and to prevent any artifact due to the anisotropic resolution of confocal microscopy, we next performed an isotropic 3D reconstruction (Fig. 3b-f) 23 . By combining over fourteen mature centrioles, we generated an isotropic averaged volume of the centriole (Fig.…”

A workflow for sizing oligomeric biomolecules based on cryo single molecule localization microscopy

“…In principle, particle averaging approaches allow for circumventing the issue of statistical distortions in SMLM. Similarly to single particle reconstruction methods used in cryo-electron microscopy (cryo-EM), hundreds to thousands of identical copies of the same particle are imaged, and subsequently combined to yield an averaged super-particle 10,11 . In case of unknown structures template-free registration methods have to be employed.…”