Measuring cryo-TEM sample thickness using reflected light microscopy and machine learning

In cryo-transmission electron microscopy (cryo-TEM), sample thickness is one of the most important parameters that governs image quality. When combining cryo-TEM with other imaging methods, such as light microscopy, measuring and controlling the sample thickness to ensure suitability of samples becomes even more critical due to the low throughput of such correlated imaging experiments. Here, we present a method to assess the sample thickness using reflected light microscopy and machine learning that can be used prior to TEM imaging of a sample. The method makes use of the thin-film interference effect that is observed when imaging narrow-band LED light sources reflected by thin samples. By training a neural network to translate such reflection images into maps of the underlying sample thickness, we are able to accurately predict the thickness of cryo-TEM samples using a light microscope. We exemplify our approach using mammalian cells grown on TEM grids, and demonstrate that the thickness predictions are highly similar to the measured sample thickness. The open-source software described herein, including the neural network and algorithms to generate training datasets, is freely available at github.com/bionanopatterning/thicknessprediction. With the recent development of in situ cellular structural biology using cryo-TEM, there is a need for fast and accurate assessment of sample thickness prior to high-resolution imaging. We anticipate that our method will improve the throughput of this assessment by providing an alternative method to screening using cryo-TEM. Furthermore, we demonstrate that our method can be incorporated into correlative imaging workflows to locate intracellular proteins at sites ideal for high-resolution cryo-TEM imaging.Microscopic imaging and technolog

One-pot synthesis of defined-length ssDNA for multiscaffold DNA origami

DNA origami nanostructures generally require a single scaffold strand of specific length, combined with many small staple strands. Ideally, the length of the scaffold strand should be dictated by the size of the designed nanostructure. However, synthesizing arbitrary-length single-stranded DNA in sufficient quantities is difficult. Here, we describe a straightforward and accessible method to produce defined-length ssDNA scaffolds using PCR and subsequent selective enzymatic digestion with T7 exonuclease. This approach produced ssDNA with higher yields than other methods and without the need for purification, which significantly decreased the time from PCR to obtaining pure DNA origami. Furthermore, this enabled us to perform true one-pot synthesis of defined-size DNA origami nanostructures. Additionally, we show that multiple smaller ssDNA scaffolds can efficiently substitute longer scaffolds in the formation of DNA origami.Microscopic imaging and technolog

scNodes: a correlation and processing toolkit for super-resolution fluorescence and electron microscopy

Correlative cryo-super resolution light and cryo-electron microscopy (SR-cryoCLEM) is an upcoming method that promises to enable accurately targeted, high-resolution in situ structure determination by combining the specificity of fluorescent labelling with the high resolution of cryoEM. As it is a novel and rapidly developing method, there is currently no dedicated open-source software available for data processing, and current implementations rely instead on numerous disjointed software packages for processing and visualization. Here, we present a solution: an open-source, standalone processing suite called ‘scNodes,’ that is specialized for (but not limited to) SR-cryoCLEM and can be used for super-resolution fluorescence data processing, image correlation, and fluorescence-guided particle picking for cryoEM.Microscopic imaging and technolog

Cryo-electron microscopy and biochemical analysis offer insights into the effects of acidic pH, such as occur during acidosis, on the complement binding properties of C-reactive protein

The pentraxin family of proteins includes C-reactive protein (CRP), a canonical marker for the acute phase inflammatory response. As compared to normal physiological conditions in human serum, under conditions associated with damage and inflammation, such as acidosis and the oxidative burst, CRP exhibits modulated biochemical properties that may have a structural basis. Here, we explore how pH and ligand binding affect the structure and biochemical properties of CRP. Cryo-electron microscopy was used to solve structures of CRP at pH 7.5 or pH 5 and in the presence or absence of the ligand phosphocholine (PCh), which yielded 7 new high-resolution structures of CRP, including pentameric and decameric complexes. Structures previously derived from crystallography were imperfect pentagons, as shown by the variable angles between each subunit, whereas pentameric CRP derived from cryoEM was found to have C5 symmetry, with subunits forming a regular pentagon with equal angles. This discrepancy indicates flexibility at the interfaces of monomers that may relate to activation of the complement system by the C1 complex. CRP also appears to readily decamerise in solution into dimers of pentamers, which obscures the postulated binding sites for C1. Subtle structural rearrangements were observed between the conditions tested, including a putative change in histidine protonation that may prime the disulphide bridges for reduction and enhanced ability to activate the immune system. Enzyme-linked immunosorbent assays showed that CRP had markedly increased association to the C1 complex and immunoglobulins under conditions associated with acidosis, whilst a reduction in the Ca2+ concentration lowered this pH-sensitivity for C1q, but not immunoglobulins, suggesting different modes of binding. These data suggest a model whereby a change in the ionic nature of CRP and immunological proteins can make it more adhesive to potential ligands without large structural rearrangements.Microscopic imaging and technolog

Correlated Cryo Super‐Resolution Light and Cryo‐Electron Microscopy on Mammalian Cells Expressing the Fluorescent Protein rsEGFP2

Super‐resolution light microscopy (SRM) enables imaging of biomolecules within cells with nanometer precision. Cryo‐fixation by vitrification offers optimal structure preservation of biological specimens and permits sequential cryo electron microscopy (cryoEM) on the same sample, but is rarely used for SRM due to various technical challenges and the lack of fluorophores developed for vitrified conditions. Here, a protocol to perform correlated cryoSRM and cryoEM on intact mammalian cells using fluorescent proteins and commercially available equipment is described. After cell culture and sample preparation by plunge‐freezing, cryoSRM is performed using the reversibly photoswitchable fluorescent protein rsEGFP2. Next, a super‐resolved image is reconstructed to guide cryoEM imaging to the feature of interest. Finally, the cryoSRM and cryoEM images are correlated to combine information from both imaging modalities. Using this protocol, a localization precision of 30 nm for cryoSRM is routinely achieved. No impediments to successive cryoEM imaging are detected, and the protocol is compatible with a variety of cryoEM techniques. When the optical set‐up and analysis pipeline is established, the total duration of the protocol for experienced cryoEM users is 3 days, not including cell culture. Microscopic imaging and technolog

Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology. Microscopic imaging and technolog

Searching a bitstream in linear time for the longest substring of any given density

Given an arbitrary bitstream, we consider the problem of finding the longest substring whose ratio of ones to zeroes equals a given value. The central result of this paper is an algorithm that solves this problem in linear time. The method involves (i) reformulating the problem as a constrained walk through a sparse matrix, and then (ii) developing a data structure for this sparse matrix that allows us to perform each step of the walk in amortised constant time. We also give a linear time algorithm to find the longest substring whose ratio of ones to zeroes is bounded below by a given value. Both problems have practical relevance to cryptography and bioinformatics.Comment: 22 pages, 19 figures; v2: minor edits and enhancement

Seizure epicenter depth and translaminar field potential synchrony underlie complex variations in tissue oxygenation during ictal initiation

Whether functional hyperemia during epileptic activity is adequate to meet the heightened metabolic demand of such events is controversial. Whereas some studies have demonstrated hyperoxia during ictal onsets, other work has reported transient hypoxic episodes that are spatially dependent on local surface microvasculature. Crucially, how laminar differences in ictal evolution can affect subsequent cerebrovascular responses has not been thus far investigated, and is likely significant in view of possible laminar-dependent neurovascular mechanisms and angioarchitecture. We addressed this open question using a novel multi-modal methodology enabling concurrent measurement of cortical tissue oxygenation, blood flow and hemoglobin concentration, alongside laminar recordings of neural activity, in a urethane anesthetized rat model of recurrent seizures induced by 4-aminopyridine. We reveal there to be a close relationship between seizure epicenter depth, translaminar LFP synchrony and tissue oxygenation during the early stages of recurrent seizures, whereby deep layer seizures are associated with decreased cross laminar synchrony and prolonged periods of hypoxia, and middle layer seizures are accompanied by increased cross-laminar synchrony and hyperoxia. Through comparison with functional activation by somatosensory stimulation and graded hypercapnia, we show that these seizure-related cerebrovascular responses occur in the presence of conserved neural-hemodynamic and blood flow-volume coupling. Our data provide new insights into the laminar dependency of seizure-related neurovascular responses, which may reconcile inconsistent observations of seizure-related hypoxia in the literature, and highlight a potential layer-dependent vulnerability that may contribute to the harmful effects of clinical recurrent seizures. The relevance of our findings to perfusion-related functional neuroimaging techniques in epilepsy are also discussed

Volcanic constraints on the unzipping of Africa from South America: Insights from new geochronological controls along the Angola margin

The breakup of Africa from South America is associated with the emplacement of the Paraná-Etendeka flood basalt province from around 134 Ma and the Tristan da Cunha plume. Yet many additional volcanic events occur that are younger than the main pulse of the Paraná-Etendeka and straddle the rift to drift phases of the main breakup. This contribution reports on new geochronological constraints from the Angolan part of the African Margin. Three coastal and one inland section have been sampled stretching across some 400 Km, with 39Ar/40Ar, U-Pb and Palaeontology used to provide age constraints. Ages from the new data range from ~100 to 81 Ma, with three main events (cr. 100, 91 and 82-81 Ma). Volcanic events are occurring within the Early to Late Cretaceous, along this part of the margin with a general younging towards Namibia. With the constraints of additional age information both onshore and offshore Angola, a clear younging trend at the early stages of rift to drift is recorded in the volcanic events that unzip from North to South. Similar age volcanic events are reported from the Brazilian side of the conjugate margin, and highlight the need to fully incorporate these relatively low volume volcanic pulses into the plate tectonic breakup models of the South Atlantic Margin

Structures of C1-IgG1 provide insights into how danger pattern recognition activates complement

Microscopic imaging and technolog