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Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

New Hunt’s House, Guy’s Campus, King’s College London

Low-temperature electron microscopy endeavors to provide "solidification of a biological specimen by cooling with the aim of minimal displacement of its components through the use of low temperature as a physical fixation strategy" (Steinbrecht and Zierold, Cryotechniques in biological electron microscopy. Springer-Verlag, Berlin, p 293, 1987). The intention is to maintain confidence that the tissue observed retains the morphology and dimensions of the living material while also ensuring soluble cellular components are not displaced. As applied to both scanning and transmission electron microscopy, cryo-electron microscopy is a strategy whereby the application of low-temperature techniques are used to reduce or remove processing artifacts which are commonly encountered in more conventional room temperature electron microscopy techniques which rely heavily on chemical fixation and heavy metal staining. Often, cryo-electron microscopy allows direct observation of specimens, which have not been stained or chemically fixed. 

Low-temperature electron microscopy: techniques and protocols.

The interplay between porosity and electromigration can be used to manipulate atoms resulting in mass fabrication of nanoscale structures. Electromigration usually results in the accumulation of atoms accompanied by protrusions at the anode and atomic depletion causing voids at the cathode. Here we show that in porous media the pattern of atomic deposition and depletion is altered such that atomic accumulation occurs over the whole surface and not just at the anode. The effect is explained by the interaction between atomic drift due to electric current and local temperature gradients resulting from intense Joule heating at constrictions between grains. Utilizing this effect, a porous silver substrate is used to mass produce free-standing silver nanorods with very high aspect ratios of more than 200 using current densities of the order of 10(8) A/m(2). This simple method results in reproducible formation of shaped nanorods, with independent control over their density and length. Consequently, complex patterns of high quality single crystal nanorods can be formed in-situ with significant advantages over competing methods of nanorod formation for plasmonics, energy storage and sensing applications. 

Tunable Ultra-high Aspect Ratio Nanorod Architectures grown on Porous Substrate via Electromigration.

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes:  and  Viable and fertile single and double homozygous  and  zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However,  mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from  single mutant females showed minor chorion defects, but chorions from eggs laid by  double mutant females were more defective than those from  single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.

Roland A. Fleck

Centre for Ultrastructural Imaging

New Hunt’s House, Guy’s Campus, King’s College London

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Roland A. Fleck

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New Hunt’s House, Guy’s Campus, King’s College London

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Centre for Ultrastructural Imaging