The effect of secondary electrons on radiolysis as observed by in liquid TEM: The role of window material and electrical bias

The effect of window material on electron beam induced phenomena in liquid phase electron microscopy (LPEM) is an interesting yet under-explored subject. We have studied the differences of electron beam induced gold nanoparticle (AuNP) growth subject to three encapsulation materials: Silicon Nitride (Si3N4), carbon and formvar. We find Si3N4 liquid cells (LCs) to result in significantly higher AuNP growth yield as compared to LCs employing the other two materials. In all cases, an electrical bias of the entire LC structures significantly affected particle growth. We demonstrate an inverse correlation of the AuNP growth rate with secondary electron (SE) emission from the windows. We attribute these differences at least in part to variations in SE emission dynamics, which is seen as a combination of material and bias dependent SE escape flux (SEEF) and SE return flux (SERF). Furthermore, our model predictions qualitatively match electrochemistry expectations

Serial protein crystallography in an electron microscope

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

Fibroblast growth factor signalling controls nervous system patterning and pigment cell formation in Ciona intestinalis

During the development of the central nervous system (CNS), combinations of transcription factors and signalling molecules orchestrate patterning, specification and differentiation of neural cell types. In vertebrates, three types of melanin-containing pigment cells, exert a variety of functional roles including visual perception. Here we analysed the mechanisms underlying pigment cell specification within the CNS of a simple chordate, the ascidian Ciona intestinalis. Ciona tadpole larvae exhibit a basic chordate body plan characterized by a small number of neural cells. We employed lineage-specific transcription profiling to characterize the expression of genes downstream of fibroblast growth factor signalling, which govern pigment cell formation. We demonstrate that FGF signalling sequentially imposes a pigment cell identity at the expense of anterior neural fates. We identify FGF-dependent and pigment cell-specific factors, including the small GTPase, Rab32/38 and demonstrated its requirement for the pigmentation of larval sensory organs

Influence of the beam and window material on liquid phase transmission electron microscopy

Over the last two decades, liquid phase electron microscopy (LPEM) has been a method used by Chemistry, Biology, and Physics researchers to elucidate the structure, function, and dynamics of samples in their native environment. Mainly it is used for inorganic and organic samples, nanocrystals, soft materials (polymers, gels, proteins, biological molecules), and energy storage processes. Significant advancements involve tailoring the liquid cell (LC) support structure (window dimensions or material) and imaging parameters (dose rate, beam energy, or imaging modality- STEM vs. TEM). However, the following limitations remain: bulging of the window material, thick liquid layers, poor contrast and resolution for low scattering specimens, expensive fabrication steps (time and cost), and beam-induced sample damage or movement. Therefore, this work aims to characterize the influence of the electron beam and window architectures (material and configuration) on LPEM experiments.This thesis focuses on two areas: 1) fabricating innovative LC architectures and 2) using the nucleation and growth of gold nanoparticles (AuNP) as a probe for radiolysis and the often-overlooked secondary electrons (SE) escape and return. Three LC configurations are presented: 1) the environmental liquid cell (ELC), which uses amorphous silicon nitride (Si3N4) windows, a 10 μm spacer, plus pressure and flow to create thin liquid layers. 2) Static 3 mm TEM grid LCs, from commercial carbon grids and for the first time from formvar coated grids. 3) Multi-well LCs with anodic aluminum oxide (AAO) as a spacer and instead of the previously published graphene windows: Si3N4), carbon, and formvar. The results from the fabrication of novel LC architectures present ways to control the liquid layer, decrease window bulging, decrease cost, and improve accessibility. The AAO LCs were used to investigate the beam’s interaction with the liquid and sample. The LC combinations revealed the dose rate to increase the AuNP growth. The AAO LCs act as individual nano-chambers. This was shown through graphene coated Si3N4) window resulting in bubble generation only within the AAO wells. These results indicated that factors besides liquid layer thickness, generation of radical species, and dose rate must influence the AuNP growth.Within LPEM, SEs are mainly considered as the cause of Si3N4) window charging. Whereas, what is often missed is that SEs are the precursor of the strongest reducing species, hydrated (aqueous) electrons (e-aq). Therefore, the growth of AuNP was used as a gauge for SE generation with formvar, carbon, and Si3N4) AAO LCs. Decreased window conductivity (Si3N4)) resulted in increased AuNP growth. Applying an electrical bias of +20 V increased and -20 V decreased the AuNP growth. The AuNP growth correlated inversely to the SE emission from the windows. A model describing the influence of window material and bias depicting the SE escape flux (SEEF) and SE return flux (SERF) was introduced that qualitatively matched simulations and electrochemical expectations. Overall, the results from the total work demonstrate the importance of the window material and imaging parameters on the interaction of the electron beam with the solution, sample, and window. Future experiments can use the results presented here to mitigate issues surrounding LPEM.In den letzten zwei Jahrzehnten wurde die Flüssigphasen-Elektronenmikroskopie (LPEM) von Forschern aus den Bereichen Chemie, Biologie und Physik eingesetzt, um die Struktur, Funktion und Dynamik von Proben in ihrer natürlichen Umgebung zu erforschen. Hauptsächlich wird sie für anorganische und organische Proben, Nanokristalle, weiche Materialien (Polymere, Gele, Proteine, biologische Moleküle) und Energiespeicherprozesse eingesetzt. Zu den wesentlichen Fortschritten gehört die Anpassung der Trägerstruktur der Flüssigkeitszelle (Fenstergröße oder Material) und der Bildgebungsparameter (Dosisleistung, Strahlenergie oder Bildgebungsmodalität - STEM vs. TEM). Die folgenden Einschränkungen bleiben jedoch bestehen: Ausbeulung des Fenstermaterials, dicke Flüssigkeitsschichten, schlechter Kontrast und Auflösung bei Proben mit geringer Streuung, teure Herstellungsschritte (Zeit und Kosten) und strahleninduzierte Probenschäden oder-bewegungen. Daher zielt diese Arbeit darauf ab, den Einfluss des Elektronenstrahls und der Fensterarchitekturen (Material und Konfiguration) auf LPEM-Experimente grundlegend zu charakterisieren.Diese Arbeit konzentriert sich auf zwei Bereiche: 1) die Herstellung innovativer LC-Architekturen und 2) die Nutzung der Keimbildung und des Wachstums von Goldnanopartikeln (AuNP) als Sonde für die Radiolyse und das oft übersehene Entweichen und Zurückkehren von Sekundärelektronen. Es werden drei LC-Konfigurationen vorgestellt: 1) die Environmental Liquid Cell (ELC), die Fenster aus Siliziumnitrid (Si3N4), einen 10 μm großen Abstandshalter sowie Druck und Strömung zur Erzeugung dünner Flüssigkeitsschichten verwendet. 2) Statische 3 mm TEM-Gitter-LCs aus handelsüblichen Kohlenstoffgittern und zum ersten Mal aus formvar-beschichteten Gittern. 3) Multiwell-LCs mit anodischem Aluminiumoxid (AAO) als Abstandshalter und anstelle der zuvor veröffentlichten Graphenfenster: Si3N4, Kohlenstoff und Formvar. Die Ergebnisse der Herstellung neuartiger LC-Architekturen zeigen Möglichkeiten zur Kontrolle der Flüssigkeitsschicht, zur Verringerung der Fensterausbuchtung, zur Kostensenkung und zur Verbesserung der Zugänglichkeit. Die AAO-LCs wurden verwendet, um die Wechselwirkung des Strahls mit der Flüssigkeit und der Probe zu untersuchen. Die LC-Kombinationen zeigten, dass die Dosisleistung das AuNP-Wachstum erhöht. Die AAO-LCs wirken als individuelle Nanokammern. Dies wurde durch ein mit Graphen beschichtetes Si3N4-Fenster gezeigt, das zu einer Blasenbildung nur innerhalb der AAO-Wells führt. Die Ergebnisse deuten darauf hin, dass neben der Dicke der Flüssigkeitsschicht, der Erzeugung von Radikalspezies und der Dosisleistung weitere Faktoren das AuNP-Wachstum beeinflussen müssen.Innerhalb von LPEM werden hauptsächlich SEs als Ursache für die Aufladung des Si3N4-Fensters angesehen. Was jedoch oft übersehen wird, ist, dass SEs der Vorläufer der stärksten reduzierenden Spezies, hydrated (aqueous) electrons (e-aq), sind. Daher wurde das Wachstum von AuNP als Maßstab für die Erzeugung von SE mit formvar, Kohlenstoff und Si3N4 AAO LCs. Eine verringerte Fensterleitfähigkeit (Si3N4) führte zu einem erhöhten AuNP-Wachstum. Eine elektrische Vorspannung von +20 V erhöhte und -20 V verringerte das AuNP-Wachstum. Das AuNP-Wachstum korrelierte umgekehrt mit der SE-Emission von den Fenstern. Es wurde ein Modell vorgestellt, das den Einfluss des Fenstermaterials und der Vorspannung beschreibt, das den SE escape flux (SEEF) und SE return flux (SERF) darstellt und qualitativ mit den Simulationen und elektrochemischen Erwartungen übereinstimmt. Insgesamt zeigen die Ergebnisse der gesamten Arbeit, wie wichtig das Fenstermaterial und die Abbildungsparameter für die Wechselwirkung des Elektronenstrahls mit der Lösung, der Probe und dem Fenster sind. Zukünftige Experimente können die hier vorgestellten Ergebnisse nutzen, um Probleme im Zusammenhang mit LPEM zu entschärfen

Influence of the beam and window material on liquid phase transmission electron microscopy

Over the last two decades, liquid phase electron microscopy (LPEM) has been a method used by Chemistry, Biology, and Physics researchers to elucidate the structure, function, and dynamics of samples in their native environment. Mainly it is used for inorganic and organic samples, nanocrystals, soft materials (polymers, gels, proteins, biological molecules), and energy storage processes. Significant advancements involve tailoring the liquid cell (LC) support structure (window dimensions or material) and imaging parameters (dose rate, beam energy, or imaging modality- STEM vs. TEM). However, the following limitations remain: bulging of the window material, thick liquid layers, poor contrast and resolution for low scattering specimens, expensive fabrication steps (time and cost), and beam-induced sample damage or movement. Therefore, this work aims to characterize the influence of the electron beam and window architectures (material and configuration) on LPEM experiments. This thesis focuses on two areas: 1) fabricating innovative LC architectures and 2) using the nucleation and growth of gold nanoparticles (AuNP) as a probe for radiolysis and the often-overlooked secondary electrons (SE) escape and return. Three LC configurations are presented: 1) the environmental liquid cell (ELC), which uses amorphous silicon nitride (Si3N4) windows, a 10 μm spacer, plus pressure and flow to create thin liquid layers. 2) Static 3 mm TEM grid LCs, from commercial carbon grids and for the first time from formvar coated grids. 3) Multi-well LCs with anodic aluminum oxide (AAO) as a spacer and instead of the previously published graphene windows: Si3N4), carbon, and formvar. The results from the fabrication of novel LC architectures present ways to control the liquid layer, decrease window bulging, decrease cost, and improve accessibility. The AAO LCs were used to investigate the beam’s interaction with the liquid and sample. The LC combinations revealed the dose rate to increase the AuNP growth. The AAO LCs act as individual nano-chambers. This was shown through graphene coated Si3N4) window resulting in bubble generation only within the AAO wells. These results indicated that factors besides liquid layer thickness, generation of radical species, and dose rate must influence the AuNP growth. Within LPEM, SEs are mainly considered as the cause of Si3N4) window charging. Whereas, what is often missed is that SEs are the precursor of the strongest reducing species, hydrated (aqueous) electrons (e-aq). Therefore, the growth of AuNP was used as a gauge for SE generation with formvar, carbon, and Si3N4) AAO LCs. Decreased window conductivity (Si3N4)) resulted in increased AuNP growth. Applying an electrical bias of +20 V increased and -20 V decreased the AuNP growth. The AuNP growth correlated inversely to the SE emission from the windows. A model describing the influence of window material and bias depicting the SE escape flux (SEEF) and SE return flux (SERF) was introduced that qualitatively matched simulations and electrochemical expectations. Overall, the results from the total work demonstrate the importance of the window material and imaging parameters on the interaction of the electron beam with the solution, sample, and window. Future experiments can use the results presented here to mitigate issues surrounding LPEM.In den letzten zwei Jahrzehnten wurde die Flüssigphasen-Elektronenmikroskopie (LPEM) von Forschern aus den Bereichen Chemie, Biologie und Physik eingesetzt, um die Struktur, Funktion und Dynamik von Proben in ihrer natürlichen Umgebung zu erforschen. Hauptsächlich wird sie für anorganische und organische Proben, Nanokristalle, weiche Materialien (Polymere, Gele, Proteine, biologische Moleküle) und Energiespeicherprozesse eingesetzt. Zu den wesentlichen Fortschritten gehört die Anpassung der Trägerstruktur der Flüssigkeitszelle (Fenstergröße oder Material) und der Bildgebungsparameter (Dosisleistung, Strahlenergie oder Bildgebungsmodalität - STEM vs. TEM). Die folgenden Einschränkungen bleiben jedoch bestehen: Ausbeulung des Fenstermaterials, dicke Flüssigkeitsschichten, schlechter Kontrast und Auflösung bei Proben mit geringer Streuung, teure Herstellungsschritte (Zeit und Kosten) und strahleninduzierte Probenschäden oder-bewegungen. Daher zielt diese Arbeit darauf ab, den Einfluss des Elektronenstrahls und der Fensterarchitekturen (Material und Konfiguration) auf LPEM-Experimente grundlegend zu charakterisieren. Diese Arbeit konzentriert sich auf zwei Bereiche: 1) die Herstellung innovativer LC-Architekturen und 2) die Nutzung der Keimbildung und des Wachstums von Goldnanopartikeln (AuNP) als Sonde für die Radiolyse und das oft übersehene Entweichen und Zurückkehren von Sekundärelektronen. Es werden drei LC-Konfigurationen vorgestellt: 1) die Environmental Liquid Cell (ELC), die Fenster aus Siliziumnitrid (Si3N4), einen 10 μm großen Abstandshalter sowie Druck und Strömung zur Erzeugung dünner Flüssigkeitsschichten verwendet. 2) Statische 3 mm TEM-Gitter-LCs aus handelsüblichen Kohlenstoffgittern und zum ersten Mal aus formvar-beschichteten Gittern. 3) Multiwell-LCs mit anodischem Aluminiumoxid (AAO) als Abstandshalter und anstelle der zuvor veröffentlichten Graphenfenster: Si3N4, Kohlenstoff und Formvar. Die Ergebnisse der Herstellung neuartiger LC-Architekturen zeigen Möglichkeiten zur Kontrolle der Flüssigkeitsschicht, zur Verringerung der Fensterausbuchtung, zur Kostensenkung und zur Verbesserung der Zugänglichkeit. Die AAO-LCs wurden verwendet, um die Wechselwirkung des Strahls mit der Flüssigkeit und der Probe zu untersuchen. Die LC-Kombinationen zeigten, dass die Dosisleistung das AuNP-Wachstum erhöht. Die AAO-LCs wirken als individuelle Nanokammern. Dies wurde durch ein mit Graphen beschichtetes Si3N4-Fenster gezeigt, das zu einer Blasenbildung nur innerhalb der AAO-Wells führt. Die Ergebnisse deuten darauf hin, dass neben der Dicke der Flüssigkeitsschicht, der Erzeugung von Radikalspezies und der Dosisleistung weitere Faktoren das AuNP-Wachstum beeinflussen müssen. Innerhalb von LPEM werden hauptsächlich SEs als Ursache für die Aufladung des Si3N4-Fensters angesehen. Was jedoch oft übersehen wird, ist, dass SEs der Vorläufer der stärksten reduzierenden Spezies, hydrated (aqueous) electrons (e-aq), sind. Daher wurde das Wachstum von AuNP als Maßstab für die Erzeugung von SE mit formvar, Kohlenstoff und Si3N4 AAO LCs. Eine verringerte Fensterleitfähigkeit (Si3N4) führte zu einem erhöhten AuNP-Wachstum. Eine elektrische Vorspannung von +20 V erhöhte und -20 V verringerte das AuNP-Wachstum. Das AuNP-Wachstum korrelierte umgekehrt mit der SE-Emission von den Fenstern. Es wurde ein Modell vorgestellt, das den Einfluss des Fenstermaterials und der Vorspannung beschreibt, das den SE escape flux (SEEF) und SE return flux (SERF) darstellt und qualitativ mit den Simulationen und elektrochemischen Erwartungen übereinstimmt. Insgesamt zeigen die Ergebnisse der gesamten Arbeit, wie wichtig das Fenstermaterial und die Abbildungsparameter für die Wechselwirkung des Elektronenstrahls mit der Lösung, der Probe und dem Fenster sind. Zukünftige Experimente können die hier vorgestellten Ergebnisse nutzen, um Probleme im Zusammenhang mit LPEM zu entschärfen

Environmental drivers of plant community composition in subalpine and alpine fens of the San Juan Mountains, Colorado, USA

2015 Spring.Includes bibliographical references.Fens are a widely distributed type of wetland worldwide and offer vital habitat for plant and animal species in the Rocky Mountains. Fens support a high biodiversity of flora and fauna given the proportionally small space they occupy on the landscape, often serving as refugia for disjunct plant species at the extremes of their ranges. While some literature exists on subalpine fens in the southern Rocky Mountains of the United States, alpine fens in this region remain understudied. Alpine fens are relatively rare in the southern Rocky Mountains and are concentrated within the San Juan Mountains where topography and climate favor peat development in the alpine. While studies of montane and boreal peatlands have identified water chemistry as a main driver of plant community composition, it is unclear whether the same drivers of plant community composition are important in alpine fens in the San Juan Mountains. The goal of this study was to 1.) Describe and classify the vegetation of subalpine and alpine fens and, 2.) Determine underlying environmental variables influencing plant community composition. To do this, I mapped fens within the BLM Gunnison Management Unit (approximately 243,000 hectares). I then visited, verified, and sampled vegetation and environmental data from 33 subalpine and 32 alpine fens. To classify vegetation data into plant communities, I used hierarchical cluster analysis. I used non-metric multidimensional scaling and comparisons of ranked environmental and vegetation distance matrices to investigate relationships between plant community composition and environmental variables. I compared the influence of environmental variables on subalpine and alpine plant community composition with cumulative r² values from linear regressions with NMS axes and Spearman rank correlations between ranked vegetation and environmental distance matrices. I classified 226 stands of vegetation into 11 plant communities that were correlated with elevation and water chemistry variables. Water chemistry variables, particularly pH, EC, and bicarbonate, were more important in structuring vegetation in subalpine than alpine fens. This was in part due to a lower range in values of alpine water chemistry variables. However, lower variance in water chemistry variables did not correspond to decreased plant community diversity in the alpine. To thoroughly explain alpine fen plant community diversity, future studies should consider measuring additional variables, such as soil temperature and temporal variation in water table. Elevation was a relatively important explanatory variable for plant community composition in alpine fens, suggesting that climatic variables are important influences on community composition. Results of this research indicate that the relative importance of environmental variables differs for alpine and subalpine fen plant communities. Thus, future studies examining mountain fen plant community composition should treat alpine and subalpine fen data separately

Crystal structure and biochemical properties of a novel thermostable esterase containing an immunoglobulin-like domain

Comparative analysis of the genome of the hyperthermophilic bacterium Thermotoga maritima revealed a hypothetical protein (EstA) with typical esterase features. The EstA protein was functionally produced in Escherichia coli and purified to homogeneity. It indeed displayed esterase activity with optima at or above 95 degrees C and at pH 8.5, with a preference for esters with short acyl chains (C2-C10). Its 2.6-A-resolution crystal structure revealed a classical alpha/beta hydrolase domain with a catalytic triad consisting of a serine, an aspartate, and a histidine. EstA is irreversibly inhibited by the organophosphate paraoxon. A 3.0-A-resolution structure confirmed that this inhibitor binds covalently to the catalytic serine residue of EstA. Remarkably, the structure also revealed the presence of an N-terminal immunoglobulin (Ig)-like domain, which is unprecedented among esterases. EstA forms a hexamer both in the crystal and in solution. Electron microscopy showed that the hexamer in solution is identical with the hexamer in the crystal, which is formed by two trimers, with the N-terminal domains facing each other. Mutational studies confirmed that residues Phe89, Phe112, Phe116, Phe246, and Trp377 affect enzyme activity. A truncated mutant of EstA, in which the Ig-like domain was removed, showed only 5% of wild-type activity, had lower thermostability, and failed to form hexamers. These data suggest that the Ig-like domain plays an important role in the enzyme multimerization and activity of Est