Search

Recent advances in scanning transmission electron microscopy (STEM) have rekindled interest in multi-channel detectors and prompted the exploration of unconventional scan patterns. These emerging needs are not yet addressed by standard commercial hardware. The system described here incorporates a flexible scan generator that enables exploration of low-acceleration scan patterns, while data are recorded by a scalable eight-channel array of nonmultiplexed analog-to-digital converters. System integration with SerialEM provides a flexible route for automated acquisition protocols including tomography. Using a solid-state quadrant detector with additional annular rings, we explore the generation and detection of various STEM contrast modes. Through-focus bright-field scans relate to phase contrast, similarly to wide-field TEM. More strikingly, comparing images acquired from different off-axis detector elements reveals lateral shifts dependent on defocus. Compensation of this parallax effect leads to decomposition of integrated differential phase contrast (iDPC) to separable contributions relating to projected electric potential and to defocus. Thus, a single scan provides both a computationally refocused phase contrast image and a second image in which the signed intensity, bright or dark, represents the degree of defocus.

Cryo-electron tomography (cryo-ET) is the prime method for cell biological studies in three dimensions (3D) at high resolution. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access larger 3D volumes, on the scale of 1 micron, making it ideal to study organelles and their interactionsin situ. Here we introduce two relevant advances: a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy (cryo-CLEM). Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to isotropic resolution in the reconstruction without averaging. We show individual protein densities in a mitochondrion-ER contact in a cell region 850 nm thick. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell.

Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. X-ray powder diffraction has established that the unit cell contains a cyclic hematin dimer, yet the pro-chiral nature of heme supports formation of four distinct stereoisomers, two centrosymmetric and two chiral enantiomers. Here we apply emerging methods of in situ cryo-electron tomography and diffraction to obtain a definitive structure of biogenic hemozoin. Individual crystals take a striking polar morphology. Diffraction analysis, supported by density functional theory, indicates a compositional mixture of one centrosymmetric and one chiral dimer, whose absolute configuration has been determined on the basis of crystal morphology and interaction with the aqueous medium. Structural modeling of the heme detoxification protein suggests a mechanism for dimer selection. The refined structure of hemozoin should serve as a guide to new drug development.

ChemMedChem 16(10), 1515 - 1532 (2021). doi:10.1002/cmdc.202000895, The biogenic formation of hemozoin crystals, a crucial process in heme detoxification by the malaria parasite, is reviewed as an antimalarial drug target. We first focus on the in-vivo formation of hemozoin. A model is presented, based on native-contrast 3D imaging obtained by X-ray and electron microscopy, that hemozoin nucleates at the inner membrane leaflet of the parasitic digestive vacuole, and grows in the adjacent aqueous medium. Having observed quantities of hemoglobin and hemozoin in the digestive vacuole, we present a model that heme liberation from hemoglobin and hemozoin formation is an assembly-line process. The crystallization is preceded by reaction between heme monomers yielding hematin dimers involving fewer types of isomers than in synthetic hemozoin; this is indicative of protein-induced dimerization. Models of antimalarial drugs binding onto hemozoin surfaces are reviewed. This is followed by a description of bromoquine, a chloroquine drug analogue, capping a significant fraction of hemozoin surfaces within the digestive vacuole and accumulation of the drug, presumably a bromoquine���hematin complex, at the vacuole's membrane., Published by Wiley-VCH, Weinheim [u.a.]

We find significant differences between degradation and healing at the surface or in the bulk for each of the different APbBr3 single crystals (A = CH3NH3+, methylammonium (MA); HC(NH2)2+, formamidinium (FA); and cesium, Cs+). Using 1- and 2-photon microscopy and photobleaching we conclude that kinetics dominate the surface and thermodynamics the bulk stability. Fluorescence-lifetime imaging microscopy, as well as results from several other methods, relate the (damaged) state of the halide perovskite (HaP) after photobleaching to its modified optical and electronic properties. The A cation type strongly influences both the kinetics and the thermodynamics of recovery and degradation: FA heals best the bulk material with faster self-healing; Cs+ protects the surface best, being the least volatile of the A cations and possibly through O-passivation; MA passivates defects via methylamine from photo-dissociation, which binds to Pb2+. DFT simulations provide insight into the passivating role of MA, and also indicate the importance of the Br3- defect as well as predicts its stability. The occurrence and rate of self-healing are suggested to explain the low effective defect density in the HaPs and through this, their excellent performance. These results rationalize the use of mixed A-cation materials for optimizing both solar cell stability and overall performance of HaP-based devices, and provide a basis for designing new HaP variants. published

The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.

Malaria is a potentially fatal infectious disease caused by the obligate intracellular parasite Plasmodium falciparum. The parasite infects human red blood cells (RBC) and derives nutrition by catabolism of hemoglobin. As amino acids are assimilated from the protein component, the toxic heme is released. Molecular heme is detoxified by rapid sequestration to physiologically insoluble hemozoin crystals within the parasite’s digestive vacuole (DV). Common antimalarial drugs interfere with this crystallization process, leaving the parasites vulnerable to the by-product of their own metabolism. A fundamental debate with important implications on drug mechanism regards the chemical environment of crystallization in vivo, whether aqueous or lipid. This issue had been addressed previously by cryogenic soft X-ray tomography. We employ cryo-scanning transmission electron tomography (CSTET) to probe parasite cells throughout the life cycle in a fully hydrated, vitrified state at higher resolution. During the acquisition of CSTET data, Bragg diffraction from the hemozoin provides a uniquely clear view of the crystal boundary at nanometer resolution. No intermediate medium, such as a lipid coating or shroud, could be detected surrounding the crystals. The present study describes a unique application of CSTET in the study of malaria. The findings can be extended to evaluate new drug candidates affecting hemozoin crystal growth.

A molecular architecture is proposed for an example mitotic chromosome, human Chromosome 10. This architecture is built on a previously described interphase chromosome structure based on Cryo-EM cellular tomography (1), thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle, for mitotic chromosomes, is specific coiling of the 11-nm nucleosome fiber into large scale approximately 200 nm structures (a Slinky (2, motif cited in 3) in interphase, and then further modification and subsequent additional coiling for the final structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well known cytological configurations. In addition, proof is experimentally provided, by digital PCR technology, that G1 T-cell nuclei are diploid, thus one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils.Significance StatementThe significance of this proposed mitotic chromosome architecture is that a specific, sequenced chromosome, human Chromosome 10, can be built into a specific architecture that accounts for the dimensional values and cytological descriptions, a first time result. Since this molecular architecture is an extension of the interphase chromosome structure, a coiling of the 11-nm nucleosome fiber with further coiling, a unifying molecular structure motif is present throughout the entire mitotic cycle, interphase through mitosis.

Thick specimens, as encountered in cryo-scanning transmission electron tomography, offer special challenges to conventional reconstruction workflows. The visibility of features, including gold nanoparticles introduced as fiducial markers, varies strongly through the tilt series. As a result, tedious manual refinement may be required in order to produce a successful alignment. Information from highly tilted views must often be excluded to the detriment of axial resolution in the reconstruction. We introduce here an approach to tilt series alignment based on identification of fiducial particle clusters that transform coherently in rotation, essentially those that lie at similar depth. Clusters are identified by comparison of tilted views with a single untilted reference, rather than with adjacent tilts. The software, called ClusterAlign, proves robust to poor signal to noise ratio and varying visibility of the individual fiducials and is successful in carrying the alignment to the ends of the tilt series where other methods tend to fail. ClusterAlign may be used to generate a list of tracked fiducials, to align a tilt series, or to perform a complete 3D reconstruction. Tools to evaluate alignment error by projection matching are included. Execution involves no manual intervention, and adherence to standard file formats facilitates an interface with other software, particularly IMOD/etomo, tomo3d, and tomoalign.

This article comments on: Antreich SJ, Xiao N, Huss JC, Gierlinger N. 2021. A belt for the cell: cellulosic wall thickenings and their role in morphogenesis of the 3D puzzle cells in walnut shells. Journal of Experimental Botany 72,4744–4756.

Significance Cellular cryo-electron tomography suffers from severely compromised Z resolution due to the missing wedges of information not collected during the acquisition of tilt series. This paper shows that application of entropy-regularized deconvolution to transmission electron tomography substantially fills in this missing information, allowing for improved Z resolution and better interpretation of cellular structures., Cryo-electron tomography (cryo-ET) allows for the high-resolution visualization of biological macromolecules. However, the technique is limited by a low signal-to-noise ratio (SNR) and variance in contrast at different frequencies, as well as reduced Z resolution. Here, we applied entropy-regularized deconvolution (ER-DC) to cryo-ET data generated from transmission electron microscopy (TEM) and reconstructed using weighted back projection (WBP). We applied deconvolution to several in situ cryo-ET datasets and assessed the results by Fourier analysis and subtomogram analysis (STA).

Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-μm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [ https://en.wikipedia.org/wiki/Slinky ; motif used in Bowerman et al. , eLife 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.

It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo-CaM and holo-CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo- and holo-CaM bind to target proteins in a similar way as apo- and holo-CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in-cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in-cell components. These results show-case the importance of in-cell analyses of protein structures.

Electron microscopy (EM) is the most versatile tool for the study of matter at scales ranging from subatomic to visible. The high vacuum environment and the charged irradiation require careful stabilization of many specimens of interest. Biological samples are particularly sensitive due to their composition of light elements suspended in an aqueous medium. Early investigators developed techniques of embedding and staining with heavy metal salts for contrast enhancement. Indeed, the Nobel Prize in 1974 recognized Claude, de Duve, and Palade for establishment of the field of cell biology, largely due to their developments in separation and preservation of cellular components for electron microscopy. A decade later, cryogenic fixation was introduced. Vitrification of the water avoids the need for dehydration and provides an ideal matrix in which the organic macromolecules are suspended; the specimen represents a native state, suddenly frozen in time at temperatures below -150 °C. The low temperature maintains a low vapor pressure for the electron microscope, and the amorphous nature of the medium avoids diffraction contrast from crystalline ice. Such samples are extremely delicate, however, and cryo-EM imaging is a race for information in the face of ongoing damage by electron irradiation. Through this journey, cryo-EM enhanced the resolution scale from membranes to molecules and most recently to atoms. Cryo-EM pioneers, Dubochet, Frank, and Henderson, were awarded the Nobel Prize in 2017 for high resolution structure determination of biological macromolecules.A relatively untapped feature of cryo-EM is its preservation of composition. Nothing is added and nothing removed. Analytical spectroscopies based on electron energy loss or X-ray emission can be applied, but the very small interaction cross sections conflict with the weak exposures required to preserve sample integrity. To what extent can we interpret quantitatively the pixel intensities in images themselves? Conventional cryo-transmission electron microscopy (TEM) is limited in this respect, due to the strong dependence of the contrast transfer on defocus and the absence of contrast at low spatial frequencies.Inspiration comes largely from a different modality for cryo-tomography, using soft X-rays. Contrast depends on the difference in atomic absorption between carbon and oxygen in a region of the spectrum between their core level ionization energies, the so-called water window. Three dimensional (3D) reconstruction provides a map of the local X-ray absorption coefficient. The quantitative contrast enables the visualization of organic materials without stain and measurement of their concentration quantitatively. We asked, what aspects of the quantitative contrast might be transferred to cryo-electron microscopy?Compositional contrast is accessible in scanning transmission EM (STEM) via incoherent elastic scattering, which is sensitive to the atomic number Z. STEM can be regarded as a high energy, low angle diffraction measurement performed pixel by pixel with a weakly convergent beam. When coherent diffraction effects are absent, that is, in amorphous materials, a dark field signal measures quantitatively the flux scattered from the specimen integrated over the detector area. Learning to interpret these signals will open a new dimension in cryo-EM. This Account describes our efforts so far to introduce STEM for cryo-EM and tomography of biological specimens. We conclude with some thoughts on further developments.

Cryo-electron tomography (cryo-ET) allows for the high resolution visualization of biological macromolecules. However, the technique is limited by a low signal-to-noise ratio (SNR) and variance in contrast at different frequencies, as well as reduced Z resolution. Here, we applied entropy regularized deconvolution (ER DC) to cryo-electron tomography data generated from transmission electron microscopy (TEM) and reconstructed using weighted back projection (WBP). We applied DC to several in situ cryo-ET data sets, and assess the results by Fourier analysis and subtomogram analysis (STA).

Based on a model of protein denaturation rate limited by an entropy-related barrier, we derive a simple formula for virus inactivation time as a function of temperature. Loss of protein structure is described by two reaction coordinates: conformational disorder of the polymer and wetting by the solvent. These establish a competition between conformational entropy and hydrophobic interaction favoring random coil or globular states, respectively. Based on the Landau theory of phase transition, the resulting free energy barrier is found to decrease linearly with the temperature difference T − Tm, and the inactivation rate should scale as U to the power of T − Tm. This form recalls an accepted model of thermal damage to cells in hyperthermia. For SARS-CoV-2 the value of U in Celsius units is found to be 1.32. Although the fitting of the model to measured data is practically indistinguishable from Arrhenius law with an activation energy, the entropy barrier mechanism is more suitable and could explain the pronounced sensitivity of SARS-CoV-2 to thermal damage. Accordingly, we predict the efficacy of mild fever over a period of ∼24 h in inactivating the virus.

The complex environment of biological cells and tissues has motivated development of three dimensional imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryo-scanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles, and then in representative cryo-tomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.Significance statementElectron tomography is used to reveal the structure of cells in three dimensions. The combination with cryogenic fixation provides a snapshot in time of the living state. However, cryo-tomography normally requires very thin specimens due to image formation by conventional phase contrast transmission electron microscopy (TEM). The thickness constraint can be relaxed considerably by scanning TEM (STEM), yet three-dimensional (3D) reconstruction is still subject to artifacts inherent in the collection of data by tilted projections. We show here that deconvolution algorithms developed for fluorescence microscopy can suppress these artifacts, resulting in significant contrast enhancement. The method is demonstrated by cellular tomography of complex membrane structures, and by segmentation of chromatin into distinct, contiguous domains of heterochromatin and euchromatin at high and low density, respectively.

The three dimensional ultrastructure of cells and tissues comes to light with tomography. There is an inherent tension between representing molecular detail at the highest possible resolution, on one hand, and visualizing spatial relations between large organelles or even neighboring cells in large volumes, on the other. Together with its advantages for pristine sample preservation, cryo-tomography brings particular constraints. A major challenge has been the restriction to specimens thinner than the typical cell. New imaging modalities are now available to extend cryo-tomography to thicker specimens: cryo-scanning transmission electron tomography (CSTET), soft X-ray tomography (SXT), and serial surface imaging using the focused ion beam — scanning electron microscope (FIB-SEM). Each one offers specific advantages so the optimal choice depends on priorities among resolution, composition, and volume.

Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.

Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.

Electron cryo-tomography using the scanning transmission modality (STEM) enables 3D reconstruction of unstained, vitrified specimens as thick as 1μm or more. Contrast is related to mass/thickness and atomic number, providing quantifiable chemical characterization and mass mapping of intact prokaryotic and eukaryotic cells. Energy dispersive X-ray spectroscopy by STEM provides a simple, on-the-spot chemical identification of the elemental composition in sub-cellular organic bodies or mineral deposits. This chapter provides basic background and practical information for performing cryo-STEM tomography on vitrified biological cells.

The ESCRT machinery mediates scission of the intercellular bridge that connects two daughter cells at the end of cytokinesis. Structured illumination microscopy (SIM) and cryo-soft-X-ray tomography (cryo-SXT) have been used in recent years to study the topology of ESCRT-driven cytokinetic abscission. These studies revealed that the intercellular bridge is occupied by cortical rings and spiral-like filaments and that ESCRTs form ring-like structures in this region during abscission. In this chapter, we provide two protocols: a protocol for determining the spatial organization of specific ESCRT components at the intercellular bridge using SIM and a protocol for resolving the ultrastructural organization of cortical filaments at the intercellular bridge using cryo-SXT.

Foraminifera are marine protozoans that are widespread in oceans throughout the world. Understanding biomineralization pathways in foraminifera is particularly important because their calcitic shells are major components of global calcium carbonate production. We introduce here a novel correlative approach combining cryo-SEM, cryo-fluorescence imaging and cryo-EDS. This approach is applied to the study of ion transport processes in the benthic foraminifer genus Amphistegina. We confirm the presence of large sea water vacuoles previously identified in intact and partially decalcified Amphistegina lobifera specimens. We observed relatively small vesicles that were labelled strongly with calcein, and also identified magnesium (Mg)-rich mineral particles in the cytoplasm, as well as in the large sea water vacuoles. The combination of cryo-microscopy with elemental microanalysis and fluorescence imaging reveals new aspects of the biomineralization pathway in foraminifera which are, to date, unique in the world of biomineralization. This approach is equally applicable to the study of biomineralization pathways in other organisms.

Summary Grasses take up silicic acid from soil and deposit it in their leaves as solid silica. This mineral, comprising 1–10% of the grass dry weight, improves plants' tolerance to various stresses. The mechanisms promoting stress tolerance are mostly unknown, and even the mineralization process is poorly understood. To study leaf mineralization in sorghum (Sorghum bicolor), we followed silica deposition in epidermal silica cells by in situ charring and air-scanning electron microscopy. Our findings were correlated to the viability of silica cells tested by fluorescein diacetate staining. We compared our results to a sorghum mutant defective in root uptake of silicic acid. We showed that the leaf silicification in these plants is intact by detecting normal mineralization in leaves exposed to silicic acid. Silica cells were viable while condensing silicic acid into silica. The controlled mineral deposition was independent of water evapotranspiration. Fluorescence recovery after photobleaching suggested that the forming mineral conformed to the cellulosic cell wall, leaving the cytoplasm well connected to neighboring cells. As the silicified wall thickened, the functional cytoplasm shrunk into a very small space. These results imply that leaf silica deposition is an active, physiologically regulated process as opposed to a simple precipitation.

The realization of high-quality optoelectronic properties in halide perovskite semiconductors through low-temperature, low energy processing is unprecedented. Understanding the unique aspects of the formation chemistry of these semiconductors is a critical step toward understanding the genesis of high quality material via simple preparation procedures. The toolbox of preparation procedures for halide perovskites grows rapidly. The prototypical reaction is that between lead iodide (PbI2) and methylammonium iodide (CH3NH3I, abbr. MAI) to form the perovskite CH3NH3PbI3 (MAPbI3), which we discuss in this work. We investigate the conversion of small, single-crystalline PbI2 crystallites to MAPbI3 by two commonly used synthesis processes: reaction with MAI in solution or as a vapor. The single crystal nature of the PbI2 precursor allows definitive conclusions to be made about the relationship between the precursors and the final product, illuminating previously unobserved aspects of the reaction process. From i...

A fusion construct between Citrine (a YFP variant) and human ferritin (H-chain) was recently shown to form supramolecular assemblies of micrometer size when expressed in mammalian cells. The assembly process is driven by weak hydrophobic interactions leading to dimerization of YFP. Protein assembly could be suppressed at the gene level by mutation in the primary sequence of the construct. In this work, we describe the engineering of a self-assembly interface sensitive to redox state in the cell. Key hydrophobic residues of YFP were mutated systematically to cysteines. Supramolecular assembly of the Citrine-ferritin construct was in some cases preserved by formation of disulfide bonds in place of hydrophobic interactions. In others cases, assembly was abolished, resulting in a diffuse distribution of the expressed protein. A specific variant that remained diffuse under normally reducing intracellular conditions was found to self-assemble rapidly upon exposure to a thiol-specific oxidizing reagent.

The electron microscope has made paramount contributions to understanding the structure of biological molecules, cells, and tissues. In general, the most faithful preservation of biological specimens and other soft-organic materials is achieved through cryogenic fixation. The embedding medium is the native aqueous environment itself, immobilized in vitrified form by rapid or pressurized cooling. Until recently, imaging of such vitrified thin specimens by electron cryo-microscopy has been nearly synonymous with wide-field transmission electron microscopy (TEM). Several new approaches have entered the cryo-microscopy field, including soft x-ray imaging, serial surface imaging using focused ion beam scanning electron microscopy, phase plates, and scanning TEM (STEM). In this article, we focus on the STEM method and its adaptation to biological cryo-microscopy. Cryogenic imaging of unstained specimens by STEM introduces specific challenges. Difficulties were long considered insurmountable, and the potential advantages were underappreciated. Future developments in experimental setup and detector technologies will allow for extension of the method to thicker specimens with improved resolution and analytic capabilities.

Electron cryo-tomography using the scanning transmission modality (STEM) enables 3D reconstruction of unstained, vitrified specimens as thick as 1μm or more. Contrast is related to mass/thickness and atomic number, providing quantifiable chemical characterization and mass mapping of intact prokaryotic and eukaryotic cells. Energy dispersive X-ray spectroscopy by STEM provides a simple, on-the-spot chemical identification of the elemental composition in sub-cellular organic bodies or mineral deposits. This chapter provides basic background and practical information for performing cryo-STEM tomography on vitrified biological cells.

The ESCRT machinery mediates scission of the intercellular bridge that connects two daughter cells at the end of cytokinesis. Structured illumination microscopy (SIM) and cryo-soft-X-ray tomography (cryo-SXT) have been used in recent years to study the topology of ESCRT-driven cytokinetic abscission. These studies revealed that the intercellular bridge is occupied by cortical rings and spiral-like filaments and that ESCRTs form ring-like structures in this region during abscission. In this chapter, we provide two protocols: a protocol for determining the spatial organization of specific ESCRT components at the intercellular bridge using SIM and a protocol for resolving the ultrastructural organization of cortical filaments at the intercellular bridge using cryo-SXT.

Summary Bacterial cells often contain dense granules. Among these, polyphosphate bodies (PPBs) store inorganic phosphate for a variety of essential functions. Identification of PPBs has until now been accomplished by analytical methods that required drying or chemically fixing the cells. These methods entail large electron doses that are incompatible with low-dose imaging of cryogenic specimens. We show here that Scanning Transmission Electron Microscopy (STEM) of fully hydrated, intact, vitrified bacteria provides a simple means for mapping of phosphorus-containing dense granules based on quantitative sensitivity of the electron scattering to atomic number. A coarse resolution of the scattering angles distinguishes phosphorus from the abundant lighter atoms: carbon, nitrogen and oxygen. The theoretical basis is similar to Z contrast of materials science. EDX provides a positive identification of phosphorus, but importantly, the method need not involve a more severe electron dose than that required for imaging. The approach should prove useful in general for mapping of heavy elements in cryopreserved specimens when the element identity is known from the biological context. Lay description Biological cells consist primarily of the light elements: hydrogen, carbon, nitrogen, and oxygen. Heavier elements are also present in smaller quantities, e.g., calcium, magnesium, phosphorous, and iron. In certain contexts these may accumulate in specific cellular bodies or granules. While imaging in the electron microscope reveals the morphology, analytical tools are required in order to determine the elemental composition. These tools put severe constraints on sample preservation and are generally incompatible with cryogenically fixed specimens due to excessive irradiation by the electron beam. In this work we analyze phosphate-rich granules in intact, cryogenically-fixed bacteria using scanning transmission electron microscopy (STEM). Focused electrons are scattered to a range of angles that depends on the elements present locally in the specimen. We present both a theoretical basis to configure the microscope for elemental sensitivity and the experimental demonstration. In principle this analytical mapping entails the same electron radiation exposure as that required to form the image. The approach will be generally applicable for mapping of elements whose identity is known from the biological context.

Cryo-tomography of intact, vitrified cells provides a three dimensional view of their structure and organization in a snapshot of the living state. Lacking heavy metal stains, tilt series images are typically produced by defocus phase contrast. Recently, a number of other methods have been introduced for 3D cryo-imaging. These include phase plate imaging, soft X-ray tomography, serial surface imaging using the focused ion beam-scanning electron microscope, and cryo-STEM tomography (CSTET). Here we explain the basis of the STEM setup and demonstrate the capabilities of CSTET to study unfixed, fully hydrated mammalian cells. Numerous cellular features are recognized in CSTET reconstructions, including membranes, vesicles, cytoskeleton, extracellular matrix, coated pits, and ribosomes. STEM signal acquisition configuration is more flexible than defocus phase contrast, and it imposes a much less severe spatial filter on the original images. Because low spatial frequency information is retained, the STEM tomographic reconstruction more faithfully represents the mass density distribution in the specimen.

STEM modality provides major advantages for electron tomography of thicker (>300 nm) biological specimens, both for plastic-embedded, heavy-metal stained samples, and for vitrified, unstained cells. With the proliferation of modern TEM microscopes that allow for switching between TEM and STEM modes with relative ease, we expect the use of STEM tomography to increase. The concepts for STEM imaging are significantly different than for TEM, and therefore we will describe in detail the STEM imaging modality, followed by STEM tomography concepts and applications.

Electron tomography provides a detailed view into the 3D structure of biological cells and tissues. Physical fixation by vitrification of the aqueous medium provides the most faithful preservation of biological specimens in the native, fully hydrated state. Cryo-microscopy is challenging, however, because of the sensitivity to electron irradiation and due to the weak electron scattering of organic material. Tomography is even more challenging because of the dependence on multiple exposures of the same area. Tomographic imaging is typically performed in wide-field transmission electron microscopy (TEM) mode with phase contrast generated by defocus. Scanning transmission electron microscopy (STEM) is an alternative mode based on detection of scattering from a focused probe beam, without imaging optics following the specimen. While careful configuration of the illumination and detectors is required to generate useful contrast, STEM circumvents the major restrictions of phase contrast TEM to very thin specimens and provides a signal that is more simply interpreted in terms of local composition and density. STEM has gained popularity in recent years for materials science. The extension of STEM to cryomicroscopy and tomography of cells and macromolecules is summarized herein.

The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling.

Self-healing, where a modification in some parameter is reversed with time without any external intervention, is one of the particularly interesting properties of halide perovskites. While there are a number of studies showing such self-healing in perovskites, they all are carried out on thin films, where the interface between the perovskite and another phase (including the ambient) is often a dominating and interfering factor in the process. Here, self-healing in perovskite (methylammonium, formamidinium, and cesium lead bromide (MAPbBr

Plasmodium falciparum, the causative agent of the deadliest form of human malaria, alternates expression of variable antigens, encoded by members of a multi-copy gene family named var. In var2csa, the var gene implicated in pregnancy-associated malaria, translational repression is regulated by a unique upstream open reading frame (uORF) found only in its 5′ UTR. Here, we report that this translated uORF significantly alters both transcription and posttranslational protein trafficking. The parasite can alter a protein's destination without any modifications to the protein itself, but instead by an element within the 5′ UTR of the transcript. This uORF-dependent localization was confirmed by single molecule STORM imaging, followed by fusion of the uORF to a reporter gene which changes its cellular localization from cytoplasmic to ER-associated. These data point towards a novel regulatory role of uORF in protein trafficking, with important implications for the pathology of pregnancy-associated malaria.

Summary Huntington’s disease is caused by an abnormally long polyglutamine tract in the huntingtin protein. This leads to the generation and deposition of N-terminal exon1 fragments of the protein in intracellular aggregates. We combined electron tomography and quantitative fluorescence microscopy to analyze the structural and material properties of huntingtin exon1 assemblies in mammalian cells, in yeast, and in vitro. We found that huntingtin exon1 proteins can form reversible liquid-like assemblies, a process driven by huntingtin’s polyQ tract and proline-rich region. In cells and in vitro, the liquid-like assemblies converted to solid-like assemblies with a fibrillar structure. Intracellular phase transitions of polyglutamine proteins could play a role in initiating irreversible pathological aggregation., Graphical Abstract, Highlights • Aggregates of huntingtin exon1 exist in distinct liquid-like and solid-like forms • Liquid-like assembly formation is driven by polyQ and proline-rich regions of exon1 • The liquid-like assemblies convert into solid-like assemblies in vitro and in cells • Electron tomography reveals liquid and solid assemblies have distinct structures, In Huntington’s disease, a polyglutamine expanded exon1 fragment of the huntingtin protein forms aggregates in the brains of affected individuals. Peskett et al. show that this protein fragment can form reversible liquid-like assemblies, which convert into solid-like fibrillar assemblies when the polyglutamine tract reaches disease-associated lengths.

Cryo-electron tomography (CET) of fully hydrated, vitrified biological specimens has emerged as a vital tool for biological research. For cellular studies, the conventional imaging modality of transmission electron microscopy places stringent constraints on sample thickness because of its dependence on phase coherence for contrast generation. Here we demonstrate the feasibility of using scanning transmission electron microscopy for cryo-tomography of unstained vitrified specimens (CSTET). We compare CSTET and CET for the imaging of whole bacteria and human tissue culture cells, finding favorable contrast and detail in the CSTET reconstructions. Particularly at high sample tilts, the CSTET signals contain more informative data than energy-filtered CET phase contrast images, resulting in improved depth resolution. Careful control over dose delivery permits relatively high cumulative exposures before the onset of observable beam damage. The increase in acceptable specimen thickness broadens the applicability of electron cryo-tomography.