|AVS 57th International Symposium & Exhibition|
|Applied Surface Science||Wednesday Sessions|
|Session:||Surface Mass Spectrometry: SIMS and Beyond|
|Presenter:||M.A. Robinson, University of Washington|
|Authors:||M.A. Robinson, University of Washington
D.G. Castner, University of Washington
|Correspondent:||Click to Email|
Over the last decade time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been increasingly used for examining (imaging) biological samples ranging from surface immobilized proteins to tissue sections to single cells. Due to the ultra high vacuum requirement of the ToF-SIMS technique careful and stringent sample preparation protocols are necessary to preserve the native state of these samples. There are a variety of preparation procedures that have been developed to address this need. However, there is still debate as to which procedure best preserves cell chemistry and morphology. The goal of this study is to further elucidate the differences in information that can be obtained when comparing cellular samples that have been plunge cooled and then freeze dried versus those that have been plunge cooled and analyzed in the frozen hydrated state.
Freeze drying biological samples after cryofixation is a much simpler method of sample preparation compared to frozen hydrated. We have shown that freeze drying cryofixed samples preserves the native morphology as well as the K+/Na+ gradient in single NIH 3T3 fibroblasts, as shown in Figures 1a and 1b. Given optimal preparation procedures, images of subcellular compartments can be realized, as seen in Figure 1c. A single cell is shown after C60++ sputtering (C60++ ion dose = 4.3x1014 ions/cm2). The nucleus can be seen in the circular area within the cell that lacks C3H8N+ signal. A nucleolus is observed as the bright spot within the nucleus. Also noticeable is the C3H8N+ signal is the Golgi apparatus in the perinuclear region.
The question that remains is what type of information can be obtained from samples that have been freeze dried? Some groups have reported that freeze drying will cause cellular damage , while others have reported detrimental effects from frozen hydrated analysis . Freeze drying continues to be the most popular preparation method after cryofixation, most likely because it is less complex than frozen hydrated analysis, as well as the fact that few ToF-SIMS instruments are setup to easily perform this type of investigation. We are adapting a liquid nitrogen cooled sample stage to allow for the rapid entry of cellular samples at liquid nitrogen temperatures to avoid sample warming and ice crystal formation. Ice crystal formation must be avoided in frozen hydrated analysis of biological material, as ice crystal formation will rupture cell membranes causing a loss of morphological and chemical information.
The results from this study shows the differences in information obtained when analyzing similar cellular samples in the freeze dried state versus the frozen hydrated state.