To better preserve ultrastructure, we propose to purchase a fully automated Leica high- pressure freezer and an automated freeze-substitution system. For thick, dense specimens, standard fixatives may not penetrate quickly enough to accurately preserve delicate membrane and proteinaceous structures, nor capture the highly dynamic changes characteristic of many biological systems. Preliminary data directly demonstrates the need for high-pressure freezing to avoid the sensitivity of certain tissues to fixation artifacts. By automating freezing of samples, the Leica system also facilitates correlative imaging with light or fluorescence microscopy. As the largest core microscope facility on campus, our well-trained staff and established resources already serves over 250 investigator users annually. We already provide all preparative equipment and services for multiple electron microscopes. This proposed instrumentation complements our existing electron microscopy services and provides unique capabilities for correlative light microscopy with our other confocal/multiphoton microscopes. Because no equivalent instrument is available for a 55-mile radius, many time-sensitive studies proposed by major users would have been precluded by the extreme delicacy of frozen samples. As our large group of EM users (>100 investigators) learn of the advantages of high-pressure freezing, we expect this instrumentation to play a critical role for many more NIH-funded projects.
|Chung, Seyeon; Andrew, Deborah J (2014) Cadherin 99C regulates apical expansion and cell rearrangement during epithelial tube elongation. Development 141:1950-60|