The use of gold cluster focused ion beams produced by a liquid metal ion gun in a TOF-SIMS mass spectrometer is shown to dramatically enhance secondary ion emission of phospholipids and peptides. The method has been successfully tested with cells grown onto plastic slips and with mouse brain slices, without any treatment of the samples. Very reliable time-of-flight mass spectra are acquired with a low primary ion dose of a few 10(7) ions, and high lateral resolution molecular ion images are obtained for heavy ions of great biological interest. This approach offers new opportunities in pharmacological and biological research fields by localizing compounds of interest such as drugs or metabolites in tissues.
Experimental approaches for analyzing the chemical composition of animal cells with spatial resolution are important for many fields of biomedical research. The analysis of threedimensional microstructures by time-of-flight secondary-ion mass spectrometry (TOF-SIMS) is an emerging technique to make the molecular architecture of biological samples accessible. In SIMS the sample surface is bombarded by primary ions. A fraction of the energy transported in the socalled collision cascade is directed back to the sample surface and causes the desorption of neutral and charged chemical species (secondary ions) from the uppermost molecular layer. These are subsequently collected and analyzed with respect to their mass/charge ratio.[1] Today, most state-of-the-art instruments for organic applications use TOF analyzers for mass determination of the desorbed secondary ions.[2] TOF-SIMS allows the detection of all elements as well as small organic molecules in parallel and has a sensitivity down to the ppm/ femtomole range.[3] Scanning the sample surface with the primary-ion beam provides a 2D image of the chemical surface composition. Moreover, prolonged ion bombardment of the sample at a constant position leads to sputter erosion. Mass analysis of the sputtered material then reveals the vertical composition of the sample.[1] The lateral distribution of organic material can be imaged with a resolution of about 150-400 nm, [4][5][6] whereas the vertical resolution in organic polymer films was shown to be better than 30 nm. [7] Application to biological samples like cells and tissues, however, has so far been hindered by the limited signal intensities obtained from organic materials and the fact that the collision cascade destroys organic molecules and, thus, molecular information. The low signal intensities in surface analysis and the loss of molecular information in sputter depth profiling have been improved by the use of polyatomic primary ions like Au 3 + and Bi 3 + . [3,8] Moreover, buckminsterfullerenes have become available as a new ion source for sputter erosion.[9] The impact of C 60 + ions was found to be less destructive to organic samples than the common sputter ions O 2 + and Cs + .[10] Even intact organic molecules survive the sputter process.[11] Thus, it was the objective of this study to reconstruct the molecular composition of animal cells in three dimensions by applying repeated cycles of SIMS analysis of the sample surface followed by sputter erosion that exposes a deeper layer of the sample to the next round of SIMS analysis (TOF-SIMS 3D microarea analysis). In a dual-beam setup Bi 3 + primary ions were used to determine the chemical composition of the surface, and C 60 + ions were used for intermittent sputter erosion. [12] Six confluent layers of normal rat kidney (NRK) cells, grown on cover slips under ordinary cell-culture conditions, were analyzed by TOF-SIMS 3D microarea analysis after the cells had been stabilized by chemical fixation. Chemical fixation is a routine procedure to preserve the struct...
It has been found that a common shipping and packaging material for commercial AFM cantilever tips, poly(dimethylsiloxane) (PDMS), causes a thin layer of silicone oil contamination on AFM cantilever tips. Due to the similarity of elemental compositions between silicone oils and AFM cantilevers (both contain silicon and oxygen), it is difficult to detect such contaminants with the widely used surface characterization technique, X-ray photoelectron spectroscopy (XPS), since XPS provides mainly elemental and short-range chemical information. However, by using static time-of-flight secondary-ion mass spectrometry (TOF-SIMS), a technique that is extremely surface-sensitive, silicone oils on AFM cantilevers can easily be identified by their molecular fragments. A simple dip cleaning procedure using a mixture of concentrated sulfuric acid and hydrogen peroxide (piranha solution) was found to be an easy and effective way to remove organic contamination, including silicone oils, from AFM cantilever tips. It has also been shown, in both XPS and TOF-SIMS spectra, that a small amount of Au is present on the tip side of AFM cantilevers. This is most likely due to thermal diffusion of Au during the deposition of Au on the back side of the cantilevers, placed there to enhance laser reflectivity in the detection system of AFM instruments. No simple dipping approach was found to remove Au contamination on the tip side without also damaging the required Au coating on the back side of the cantilevers.
The distribution of phosphocholine ions (m/z 184, m/z 86), sodium ions, and potassium ions in thyroid tumor cells was analyzed by imaging TOF-SIMS. Repeated sputtering with a C(60) (+) source and subsequent analysis with a Bi(3) (+) gun produced a series of 138 images that were stacked to make a 3D display of the chemistry of cells. Phosphocholine was seen in the plasma membrane (m/z 184) and intracellular membranes (m/z 86). The different fragmentation of the phospholipid probably reflects the chemical composition of membranes at these sites. High intensity of secondary ion signals of potassium was seen in membrane-encompassed cellular compartments. The data indicate that potassium ions are compartmentalized in thyroid tumor cells.
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