An Electron Microscopy Proteomic Organellar Preparation (EMPOP) robot was developed as a tool for high-throughput preparation of subcellular fraction samples for electron microscopic identification. It will provide a means for validation of subcellular sample purity and confirmation of protein localization needed for organellar proteomics. / The device automates all chemical and mechanical manipulations required to prepare organelles for electron microscopic examination. It has a modular, integrated design that supports automated filtration, chemical processing, delivery and embedding of up to 96 subcellular fraction samples in parallel. Subcellular fraction specimens are extremely fragile. Consequently, the system was designed as a single unit to minimize mechanical stress on the samples by integrating a core mechanism, composed of four modular plates, and seven support subsystems for: (1) cooling, (2-3) fluid handling, (4-7) positioning. Furthermore, control software was developed specifically for the system to provide standardized, reproducible sample processing while maintaining flexibility for adjustment and recall of operational parameters. / Development of the automated process progressed from initial validation experiments and process screening to define operational parameters for preservation of sample integrity and establish a basic starting point for successful sample preparation. A series of successive modifications to seal the local environment of the samples and minimize the effect of fluidic perturbations further increased process performance. Subsequent testing of the robot's full sample preparation capacity used these refinements to generate 96 samples in approximately 16 hours; reducing the time and labor requirement of equivalent manual preparation by up to 1,000 fold. / These results provide a basis for a structured approach toward process optimization and subsequent utilization the device for massive, parallel preparation of subcellular fraction samples for electron microscopic screening and quantitative analysis of subcellular and protein targets necessary for high-throughput proteomics.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.102768 |
| Date | January 2006 |
| Creators | Waterbury, Raymond. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biomedical Engineering) |
| Rights | © Raymond Waterbury, 2006 |
| Relation | alephsysno: 002541510, proquestno: AAINR27856, Theses scanned by UMI/ProQuest. |
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