Relative Quantification of Cellular Sections with Molecular Depth Profiling ToF-SIMS Imaging.

We report the use of SIMS imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C(60) (+) ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture.

Which is more important in bioimaging SIMS experiments-The sample preparation or the nature of the projectile?

Sample preparation is central to acquiring meaningful molecule-specific images with SIMS, especially when submicron lateral resolution is involved. The issue is to maintain the distribution of target molecules while attempting to introduce biological cells or tissue into the high vacuum environment of the mass spectrometer. Here we compare freeze-drying, freeze-etching, freeze-fracture and trehalose vitrification as possible strategies for these experiments. The results show that the prospects for successful imaging experiments are greatly improved with all of these methods when using cluster ion bombardment, particularly C(60) (+) ions, not only due to increased sensitivity of this projectiles, but also since it removes contamination overlayers without insult to the underlying chemistry. The emergence of 3-dimensional imaging capabilities also suggests that sample preparation should not perturb the 3-dimensional morphology of the cell, a situation not generally possible during freeze-drying. Hence, sample preparation and projectile type are strongly coupled parameters for bioimaging with mass spectrometry.