Developing affordable wet-sample electron microscopy integrated with a temperature controlled sample holder

Scanning electron microscopy (SEM) is widely used to analyze the size, shape and composition of material systems. However, using this tool for analyzing systems such as particles suspended in solution, requires drastic sample alterations, such as precipitation and fixation. Besides altering their environment, this exposes the particles to the harsh conditions within an electron microscope, such as high vacuum and electron beam exposure. To this end, the first goal of this study was to develop methodologies for imaging wet samples using electron microscopy. This is realized by creating a sandwich structure containing the solution of interest between a partially electron transparent window and a silicon substrate. The ability of the developed imaging cells to provide good imaging conditions is demonstrated with a variety of samples including polystyrene spheres, polymeric microgels and spindle shaped nanoparticles. As some of the systems investigated are temperature sensitive, the second goal of the project was to develop a temperature controlled stage that can be integrated with the SEM. In the future this heating stage will be used alongside the wet samples to image microgels above and below their critical solution temperature.https://engagedscholarship.csuohio.edu/u_poster_2015/1030/thumbnail.jp

Characterization of Microgels in Ionic Liquid

Microgels are thermoresponsive polymeric nanoparticles whose size in aqueous solution is dependent on temperature. The microgels were studied using both dynamic light scattering (DLS) and scanning electron microscopy (SEM) to better understand the nanoparticles dynamics. The first part of the study focused on developing a controlled preparation procedure which would generate reproducible SEM images on a wet sample. The ionic liquid was mixed with a dilute solution of microgels and water was dried using nitrogen gas. This technique allowed a large volume of microgels to easily transition from their natural water solvent to a low vapor pressure ionic solvent. The second part of the study attempts to correlate the diffusion found from microgels in ionic liquid using scanning electron microscopy to the statistical average diffusion measured with dynamic light scattering. The microgels in ionic liquid observed with SEM exhibited the same radius that was measured with DLS for microgels in a water based solvent.https://engagedscholarship.csuohio.edu/u_poster_2018/1033/thumbnail.jp

Deducing Shape of Anisotropic Particles in Solution from Light Scattering: Spindles and Nanorods

Depolarized Dynamic light scattering (DDLS) enables to measure in situ rotational and translational diffusion of nanoparticles suspended in solution. Their size, shape, diffusion, and intermolecular interactions can be interred then from DDLS data using various models of diffusion. Incorporating DDLS to analyze the dimensions of easily imaged elongated particles, such as Iron (III) oxyhydroxide Spindles (FeOOH) and gold coated Nanorods, will allow a deeper understanding between rotational/translational diffusion and size distribution of hard-to-image anisotropic wet systems such as micelles, microgels, and protein complexes. The emphasis of this study was to look at the aged FeOOH Spindle sample, and explore the size distribution and modeling of the Nanorod particles. The light scattering results obtained from the basic model of non-interacting prolate ellipsoids offered dimensions similar (within 15%) to the size distribution from the Scanning Electron Microscope (SEM). The results, however, were somewhat different from the original particle size possibly due to sample aging and agglomeration of the FeOOH Spindles. Conversely, the Nanorod dimensions obtained from the Prolate Ellipsoid Model differed by a factor 1.2-2 from the values obtained by Transmission Electron Microscopy and SEM. The significant difference between DDLS and imaging results is due to the nature of the modeling employed (ellipsoid was used to model cylindrically shaped particles with spherical caps).https://engagedscholarship.csuohio.edu/u_poster_2015/1032/thumbnail.jp

Phosphorylation of XIAP by CDK1-cyclin B controls mitotic cell death

Regulation of cell death is crucial for the response of cancer cells to drug treatments that cause arrest in mitosis, and is likely to be important for protection against chromosome instability in normal cells. Prolonged mitotic arrest can result in cell death by activation of caspases and the induction of apoptosis. Here, we show that Xlinked inhibitor of apoptosis (XIAP) plays a key role in the control of mitotic cell death. Ablation of XIAP expression sensitises cells to prolonged mitotic arrest caused by a microtubule poison. XIAP is stable during mitotic arrest, but its function is controlled through phosphorylation by the mitotic kinase CDK1-cyclin-B1 at S40. Mutation of S40 to a phosphomimetic residue (S40D) inhibits binding to activated effector caspases and abolishes the anti-apoptotic function of XIAP, whereas a non-phosphorylatable mutant (S40A) blocks apoptosis. By performing live-cell imaging, we show that phosphorylation of XIAP reduces the threshold for the onset of cell death in mitosis. This work illustrates that mitotic cell death is a form of apoptosis linked to the progression of mitosis through control by CDK1-cyclin-B1

Developing affordable wet-sample electron microscopy integrated with a temperature controlled sample holder

Scanning electron microscopy (SEM) is widely used to analyze the size, shape and composition of material systems. However, using this tool for analyzing systems such as particles suspended in solution, requires drastic sample alterations, such as precipitation and fixation. Besides altering their environment, this exposes the particles to the harsh conditions within an electron microscope, such as high vacuum and electron beam exposure. To this end, the first goal of this study was to develop methodologies for imaging wet samples using electron microscopy. This is realized by creating a sandwich structure containing the solution of interest between a partially electron transparent window and a silicon substrate. The ability of the developed imaging cells to provide good imaging conditions is demonstrated with a variety of samples including polystyrene spheres, polymeric microgels and spindle shaped nanoparticles. As some of the systems investigated are temperature sensitive, the second goal of the project was to develop a temperature controlled stage that can be integrated with the SEM. In the future this heating stage will be used alongside the wet samples to image microgels above and below their critical solution temperature.https://engagedscholarship.csuohio.edu/u_poster_2015/1030/thumbnail.jp

Characterization of Microgels in Ionic Liquid

Microgels are thermoresponsive polymeric nanoparticles whose size in aqueous solution is dependent on temperature. The microgels were studied using both dynamic light scattering (DLS) and scanning electron microscopy (SEM) to better understand the nanoparticles dynamics. The first part of the study focused on developing a controlled preparation procedure which would generate reproducible SEM images on a wet sample. The ionic liquid was mixed with a dilute solution of microgels and water was dried using nitrogen gas. This technique allowed a large volume of microgels to easily transition from their natural water solvent to a low vapor pressure ionic solvent. The second part of the study attempts to correlate the diffusion found from microgels in ionic liquid using scanning electron microscopy to the statistical average diffusion measured with dynamic light scattering. The microgels in ionic liquid observed with SEM exhibited the same radius that was measured with DLS for microgels in a water based solvent.https://engagedscholarship.csuohio.edu/u_poster_2018/1033/thumbnail.jp

Deducing Shape of Anisotropic Particles in Solution from Light Scattering: Spindles and Nanorods

Depolarized Dynamic light scattering (DDLS) enables to measure in situ rotational and translational diffusion of nanoparticles suspended in solution. Their size, shape, diffusion, and intermolecular interactions can be interred then from DDLS data using various models of diffusion. Incorporating DDLS to analyze the dimensions of easily imaged elongated particles, such as Iron (III) oxyhydroxide Spindles (FeOOH) and gold coated Nanorods, will allow a deeper understanding between rotational/translational diffusion and size distribution of hard-to-image anisotropic wet systems such as micelles, microgels, and protein complexes. The emphasis of this study was to look at the aged FeOOH Spindle sample, and explore the size distribution and modeling of the Nanorod particles. The light scattering results obtained from the basic model of non-interacting prolate ellipsoids offered dimensions similar (within 15%) to the size distribution from the Scanning Electron Microscope (SEM). The results, however, were somewhat different from the original particle size possibly due to sample aging and agglomeration of the FeOOH Spindles. Conversely, the Nanorod dimensions obtained from the Prolate Ellipsoid Model differed by a factor 1.2-2 from the values obtained by Transmission Electron Microscopy and SEM. The significant difference between DDLS and imaging results is due to the nature of the modeling employed (ellipsoid was used to model cylindrically shaped particles with spherical caps).https://engagedscholarship.csuohio.edu/u_poster_2015/1032/thumbnail.jp