This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Despite the many excellent studies of EM morphology after RF/FSF into acetone, samples often suffer from ice crystal damage, which is detected in the higher resolution tomographic reconstructions. While for best results the initial vitrification of samples during rapid freezing is a crucial first step, the subsequent RF/FSF process may impose its own potential for ice damage. Due to the melting temperature of acetone at -94.9?C, freeze substitution requires warming the specimen to -90?C at the initiation of the substitution process which is significantly higher than the -140o C that marks the transition between vitrified and crystalline ice [1]. What counts for pure water may not be exactly the same for a mixed protein solution as the high solute concentration of the cytoplasm may raise the phase-transition point to some extent, but likely not to reach the melting temperature of acetone. We have begun to test the merit of starting freeze substitution at lower temperatures (-140?C) with a combination of low-melting solvents in an effort to prevent ice crystal formation at the onset of freeze substitution.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000592-41
Application #
8362552
Study Section
Special Emphasis Panel (ZRG1-CB-J (40))
Project Start
2011-05-01
Project End
2012-04-30
Budget Start
2011-05-01
Budget End
2012-04-30
Support Year
41
Fiscal Year
2011
Total Cost
$21,276
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
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