Jeffrey W. Ruberti
Date of Award
Master of Science
Department or Academic Unit
College of Engineering, Department of Mechanical and Industrial Engineering
mechanical engineering, distortion, impact, liquid nitrogen, metal mirror, slam freezing, touch freezing
Introduction: Preparation of samples for electron microscopy typically balances the need to "fix" the sample against the necessary generation of artifacts which influence the interpretation of observed structures. Ultrarapid freezing by impact of a specimen with a thermally conductive metal surface, cooled to at least liquid nitrogen (LN) temperatures, has long been thought of as the gold standard for minimizing cryofixation artifact. However, there has been a long standing debate about whether or not impact with the metal surface introduces a mechanical deformation to the sample before it freezes. Though it has been argued that the mechanical deformation wave is preceded over short distances by the freezing front, no quantitative investigation has been attempted to verify this view. The principal objective of this study was to determine if impact freezing is likely to create distortion artifact. Prior to this work, there was no method, device or fixation assay which permitted an objective, controlled study of the effect of impact velocity on the fixation of a specimen. To perform the investigation (1) a recently constructed "touch" freezer was modified; (2) a dual-phase velocity profile was developed for controlling the approach of the sample to the cooled metal mirror; (3) a new assay was introduced, capable of quantifying mechanical deformation effects produced during sample impact with a cooled copper mirror; and (4) the hypothesis that mechanical distortion of the sample by impact freezing is proportional to impact velocity was tested. Methods: The distortion assay was produced using a polystyrene microsphere-infused Laponite clay-based, shear-thinning gel (90% water) which was transparent enough to be scanned with an inverted light microscope to determine pre- and postimpact bead position. Effect of impact velocity on the gel structure were examined by impact freezing the gel using a dual-phase velocity profile which initially accelerates the sample to 4.5 m s-1 to avoid premature sample cooling, and decelerates it to 2.5, 5, 10, or 30 cm s-1for the final 0.1 seconds before making contact with the mirror. Samples were z-scanned using differential interference contrast microscopy pre- and post-impact freezing. Three dimensional point clouds of the microspheres were generated from the images and analyzed using a series of custom written MATLAB algorithms. The analysis yields the strain of the 3-dimensional point cloud of the beads which reflects the effect of the impact of the gel with the mirror. Results: Strain comparisons showed an increasing trend in sample distortion with increasing impact velocity. Specifically, the 2.5, 5, and 10 cm s-1 impact velocity strains were statistically different (P = 0.011, 0.014, and 0.022) from those of the 30 cm s-1 impact velocity. Attempts to use a standard commercial slam freezer (Delaware Diamond Knives' Cryogun: impact velocity 2.3 m s-1) produced distortions so great that the point clouds could not be correlated. Conclusions: The results from this study support the hypothesis approach velocity influences sample distortion in impact freezing. Further, we have demonstrated the efficacy of a new method (dual-phase velocity profile) for impact freezing that minimizes sample distortion incurred during contact with a cooled metal surface. Finally, we have generated a new assay capable of evaluating other impact freezing designs based on the impact distortion they introduce.
Ritzakis, Nector, "Method and evaluation for minimization of mechanical effects from impact velocity for the optimization of freezing quality of metal mirror impact freezers" (2011). Mechanical Engineering Master's Theses. Paper 53. http://hdl.handle.net/2047/d20002424
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