Research Project 2: Development of 3-D Raman Confocal Microspectroscopy Techniques to Detect Molecular Mechanisms and Sites of Damage in Mammalian Cells during Freeze/Thaw

Developing new methods of preserving biospecimens and technology to improve biospecimen quality.

The main goal of this study is to determine the correlation between the biophysical state of the intracellular medium during cryoprocessing and the thermal/osmotic damage to cells.  Therefore, we are utilizing Confocal Raman Microspectroscopy to directly map the intracellular chemcial composition of single mammalian cells (as submicron states) during cryopreservation.  Our initial results indicate that:

1. Darkening of the cell cytoplasm - traditionally attributed to intracellular ice formation (IIF) - does not result from the formation of ice crystals, but from significant structural changes in cellular macromolecules and redistribution of the cellular material.
2. Cryoprotectants, water and organic materials are heterogeneously distributed within the cells.