Resolution improvement by 3D particle averaging in localization microscopy

Abstract

Inspired by recent developments in localization microscopy that applied averaging of identical particles in 2D for increasing the resolution even further, we discuss considerations for alignment (registration) methods for particles in general and for 3D in particular. We detail that traditional techniques for particle registration from cryo electron microscopy based on cross-correlation are not suitable, as the underlying image formation process is fundamentally different. We argue that only localizations, i.e. a set of coordinates with associated uncertainties, are recorded and not a continuous intensity distribution. We present a method that owes to this fact and that is inspired by the field of statistical pattern recognition. In particular we suggest to use an adapted version of the Bhattacharyya distance as a merit function for registration. We evaluate the method in simulations and demonstrate it on three-dimensional super-resolution data of Alexa 647 labelled to the Nup133 protein in the nuclear pore complex of Hela cells. From the simulations we find suggestions that for successful registration the localization uncertainty must be smaller than the distance between labeling sites on a particle. These suggestions are supported by theoretical considerations concerning the attainable resolution in localization microscopy and its scaling behavior as a function of labeling density and localization precision.

Isotropic resolution in fluorescence imaging by single particle reconstruction

“…We selected the centriole basal body [12] and the nuclear pore [13], which are large enough to be imaged by a confocal microscope. We compare our results with those of the only existing competing method [5], which reconstructs by averaging the input particles. We also extend the method of [5] by introducing a first step of independent deconvolutions with total-variation regularization (TV) of each particle before averaging.…”

“…We compare our results with those of the only existing competing method [5], which reconstructs by averaging the input particles. We also extend the method of [5] by introducing a first step of independent deconvolutions with total-variation regularization (TV) of each particle before averaging. We refer to this extension as Deconv/Average.…”

“…This procedure is inspired by the single-particle reconstruction framework used in cryo electron microscopy (cryo-EM) [4], where a particle is reconstructed from tomographic projections instead of volumetric acquisitions. To the best of our knowledge, the only similar existing work for fluorescence is the 3D averaging approach described in [5] for STORM. The selective-plane illumination microscopy technique (SPIM) acquires images of a single sample from multiple views and involves a related reconstruction problem, but it deals with a significantly lower number of views [6,7].…”

Reconstruction From Multiple Poses in Fluorescence Imaging: Proof of Concept

“…This procedure is inspired by the single-particle reconstruction framework used in cryo electron microscopy (cryo-EM) [4], where a particle is reconstructed from tomographic projections instead of volumetric acquisitions. To the best of our knowledge, the only similar existing work for fluorescence is the 3D averaging approach described in [5] for STORM imaging.…”

Super-resolution fluorescent methods: where next for super-resolution?

“…The coordinates that are obtained are typically used to render images, but more sophisticated forms of analysis can be performed based on the coordinates themselves. Broeken et al [8] describe how the coordinates from many similar particles (here, nuclear pore complexes) can be registered in 3D and averaged together to obtain higher-resolution structural information in a method analogous to the particle averaging approaches used in cryoelectron microscopy.…”