Molecular Surveillance of Viral Processes Using Silicon Nitride Membranes

Gilmore, Brian L.; Tanner, Justin R.; McKell, Allison O.; Boudreaux, Crystal E.; Dukes, Madeline J.; McDonald, Sarah; Kelly, Deborah F.

doi:10.3390/mi4010090

Cited by 5 publications

(8 citation statements)

References 22 publications

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“…Negatively stained specimens were prepared and examined using TEM. Consistent with our previous findings [11], DLPs prepared in the presence of IgG antibodies can be readily recruited to the SiN surface, however, specimens lacking the antibody coating recruited little to no DLPs (Figure 1). As SiN microchips are hydrophobic in nature, they provide an ideal substrate to promote interactions with the lipid tail groups that are exposed at the air-water interface in the Ni-NTA lipid layers.…”

Section: Cryo-sin Specimens Are Easily Imaged Using Low-dose Temsupporting

confidence: 78%

“…We produced specimens of DLPs induced to transcribe [11] implementing the same affinity capture and plunge freezing conditions that were used for inactive DLPs. Actively transcribing DLPs were prepared on functionalized holey carbon Affinity grids ( Figure 4A) and on cryo-SiN ( Figure 4B); and specimens were imaged using the same conditions described for the inactive DLPs.…”

Section: Transcribing Dlps Captured While Synthesizing Mrnamentioning

confidence: 99%

“…Affinity grids or silicon nitride microchips containing Nickelnitrilotriacetic acid (Ni-NTA) coatings were prepared as described previously [11]. The Ni-NTA functionalized lipid layers and each subsequent protein component were added to the front side of the SiN microchips.…”

Section: Affinity Grids and Cryo-sin Preparationmentioning

confidence: 99%

“…Following 1-minute incubation, the antibody solution was removed using a Hamilton syringe and purified DLPs (2-μl aliquots of 0.1 mg/ml) that were either inactive or transcriptionally active were added to the substrates for a 2-minute incubation step. Negatively stained specimens were prepared as previously described [10,11]. Frozen-hydrated specimens were prepared using a Gatan Cryoplunge™ 3 (Gatan, Inc., Pleasanton, CA) using one-sided blotting for approximately 8 seconds prior to plunging into liquid ethane.…”

Section: Affinity Grids and Cryo-sin Preparationmentioning

confidence: 99%

“…We have also recently examined rotavirus double-layered particles (DLPs) as negatively stained specimens using SiN membranes [11]. Continuing this line of investigation, here we explore the use of SiN microchips to prepare cryo-EM specimens of actively transcribing DLPs (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

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Cryo-SiN – An Alternative Substrate to Visualize Active Viral Assemblies

2013

J Analyt Molecul Tech

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Silicon nitride (SiN) membranes that are merely nanometers-thick have been introduced as new support films for electron microscopy (EM) applications. The use of these thin membranes for high-resolution imaging is widely attractive due to their consistent manufacture, flatness, and durability. Here, we utilized SiN support films to actively monitor key steps in rotavirus RNA synthesis at the nanoscale. Rotaviruses are gastrointestinal pathogens that infect pediatric populations and pose a major health problem in developing countries. We demonstrate, for the first time, that cryo-EM specimens of these viral assemblies may be prepared using SiN membranes, that we have termed "cryo-SiN". Cryo-SiN microchips containing inactive and actively transcribing rotavirus complexes were vitrified using gentleblot conditions to yield reproducibly thin ice-embedded specimens. EM imaging results indicate that cryo-SiN samples exhibited greater visual contrast than specimens prepared on conventional support films under the same conditions. The improved contrast of the cryo-SiN samples permitted us to calculate 3D structures of rotavirus complexes using a fraction of the images required for conventionally prepared specimens. We anticipate the use of cryo-SiN microchip will enable us to better investigate critical viral life-cycle processes at the molecular level under near-native conditions. AbbreviationsDouble-Layered Particles (DLPs); Silicon Nitride (SiN); Electron Microscopy (EM); Transmission Electron Microscope (TEM); Nickel-Nitrilotriacetic Acid (Ni-NTA); Nanometers (nm)

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Section: Cryo-sin Specimens Are Easily Imaged Using Low-dose Temsupporting

confidence: 78%

Section: Transcribing Dlps Captured While Synthesizing Mrnamentioning

confidence: 99%

Section: Affinity Grids and Cryo-sin Preparationmentioning

confidence: 99%

Section: Affinity Grids and Cryo-sin Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Cryo-SiN – An Alternative Substrate to Visualize Active Viral Assemblies

2013

J Analyt Molecul Tech

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Silicon Compound Nanomaterials: Exploring Emission Mechanisms and Photobiological Applications

Dutt,

Salinas,

Martínez-Tolibia

et al. 2023

Advanced Photonics Research

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After the first visible photoluminescence (PL) from porous silicon (pSi), continuous efforts are made to fabricate Si‐based compound nanomaterials embedded in matrices such as oxide, nitride, and carbide to improve optical performance and industrial acceptability. These nanomaterials’ functional and desired properties (nanoparticles and quantum dots embedded in matrices) can vary significantly when embedded in technologically relevant matrices. However, exploring the exact emission mechanisms is one of the remaining challenges from the past few decades. To cover this gap, this review discusses the morphological and optoelectronic properties of Si‐based compound nanomaterials and their correlation with the quantum confinement effect and different surface states to find precise emission mechanisms. One of the biggest challenges of using silicon nanomaterials in the biological sector is the development of sensitive materials of low/acceptable toxicity for identifying target analytes either inside/outside the biological platforms. In this scenario, silicon‐based compound matrices can offer different characteristics and advantages depending on their size configurations and PL emission mechanisms. On the other hand, a proper understanding of these multifaceted silicon nanomaterials’ optical properties (emission mechanisms) can be exploited for pathogen detection and in situ applications in cells and tissues, embarking on a new era of bioimaging technology.

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Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy

Gilmore

Varano

Dearnaley

et al. 2018

Protein Complex Assembly

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Recent advances in technology have enabled single-particle electron microscopy (EM) to rapidly progress as a preferred tool to study protein assemblies. Newly developed materials and methods present viable alternatives to traditional EM specimen preparation. Improved lipid monolayer purification reagents offer considerable flexibility, while ultrathin silicon nitride films provide superior imaging properties to the structural study of protein complexes. Here, we describe the steps for combining monolayer purification with silicon nitride microchips to create a tunable approach for the EM community.

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Molecular Surveillance of Viral Processes Using Silicon Nitride Membranes

Cited by 5 publications

References 22 publications

Cryo-SiN – An Alternative Substrate to Visualize Active Viral Assemblies

Cryo-SiN – An Alternative Substrate to Visualize Active Viral Assemblies

Silicon Compound Nanomaterials: Exploring Emission Mechanisms and Photobiological Applications

Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy

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