Elemental mapping of labelled biological specimens at intermediate energy loss in an energy-filtered TEM acquired using a direct detection device.

The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy-filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo-colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high-loss M4,5 edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N4,5 edge (excitation of 4d electrons), which occurring at a much lower energy-loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross-sections at the N4,5 edge. Acquiring EFTEM lanthanide elemental maps at the N4,5 edge instead of the M4,5 edge, provides ∼4× increase in signal-to-noise and ∼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N4,5 edge by the traditional 3-window method, is complicated due to the broad shape of the edge profile and the lower signal-above-background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second-generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln3+ per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate-loss energy-loss regime to three different cellular targets, namely using mitochondrial matrix-directed APEX2, histone H2B-Nucleosome and EdU-DNA. All the examples shown in the paper are single colour EM images only.

Robust image alignment for cryogenic transmission electron microscopy.

Cryo-electron microscopy recently experienced great improvements in structure resolution due to direct electron detectors with improved contrast and fast read-out leading to single electron counting. High frames rates enabled dose fractionation, where a long exposure is broken into a movie, permitting specimen drift to be registered and corrected. The typical approach for image registration, with high shot noise and low contrast, is multi-reference (MR) cross-correlation. Here we present the software package Zorro, which provides robust drift correction for dose fractionation by use of an intensity-normalized cross-correlation and logistic noise model to weight each cross-correlation in the MR model and filter each cross-correlation optimally. Frames are reliably registered by Zorro with low dose and defocus. Methods to evaluate performance are presented, by use of independently-evaluated even- and odd-frame stacks by trajectory comparison and Fourier ring correlation. Alignment of tiled sub-frames is also introduced, and demonstrated on an example dataset. Zorro source code is available at github.com/CINA/zorro.

Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography.

Single particle cryo-electron tomography (cryoSPT) extracts features from cryo-electron tomograms, followed by 3D classification, alignment and averaging to generate improved 3D density maps of such features. Robust methods to correct for the contrast transfer function (CTF) of the electron microscope are necessary for cryoSPT to reach its resolution potential. Many factors can make CTF correction for cryoSPT challenging, such as lack of eucentricity of the specimen stage, inherent low dose per image, specimen charging, beam-induced specimen motions, and defocus gradients resulting both from specimen tilting and from unpredictable ice thickness variations. Current CTF correction methods for cryoET make at least one of the following assumptions: that the defocus at the center of the image is the same across the images of a tiltseries, that the particles all lie at the same Z-height in the embedding ice, and/or that the specimen, the cryo-electron microscopy (cryoEM) grid and/or the carbon support are flat. These experimental conditions are not always met. We have developed a CTF correction algorithm for cryoSPT without making any of the aforementioned assumptions. We also introduce speed and accuracy improvements and a higher degree of automation to the subtomogram averaging algorithms available in EMAN2. Using motion-corrected images of isolated virus particles as a benchmark specimen, recorded with a DE20 direct detection camera, we show that our CTF correction and subtomogram alignment routines can yield subtomogram averages close to 4/5 Nyquist frequency of the detector under our experimental conditions.

Super-compression of large electron microscopy time series by deep compressive sensing learning.

The development of ultrafast detectors for electron microscopy (EM) opens a new door to exploring dynamics of nanomaterials; however, it raises grand challenges for big data processing and storage. Here, we combine deep learning and temporal compressive sensing (TCS) to propose a novel EM big data compression strategy. Specifically, TCS is employed to compress sequential EM images into a single compressed measurement; an end-to-end deep learning network is leveraged to reconstruct the original images. Owing to the significantly improved compression efficiency and built-in denoising capability of the deep learning framework over conventional JPEG compression, compressed videos with a compression ratio of up to 30 can be reconstructed with high fidelity. Using this approach, considerable encoding power, memory, and transmission bandwidth can be saved, allowing it to be deployed to existing detectors. We anticipate the proposed technique will have far-reaching applications in edge computing for EM and other imaging techniques.

The advent of structural biology in situ by single particle cryo-electron tomography.

Single particle tomography (SPT), also known as subtomogram averaging, is a powerful technique uniquely poised to address questions in structural biology that are not amenable to more traditional approaches like X-ray crystallography, nuclear magnetic resonance, and conventional cryoEM single particle analysis. Owing to its potential for in situ structural biology at subnanometer resolution, SPT has been gaining enormous momentum in the last five years and is becoming a prominent, widely used technique. This method can be applied to unambiguously determine the structures of macromolecular complexes that exhibit compositional and conformational heterogeneity, both in vitro and in situ. Here we review the development of SPT, highlighting its applications and identifying areas of ongoing development.