No abstract

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has unique capabilities in the area of high-resolution mass spectrometric imaging of biological samples. The technique offers parallel detection of native and non-native molecules at physiological concentrations with potentially submicrometer spatial resolution. Recent advances in SIMS technology have been focused on generating new ion sources that can in turn be used to eject more intact molecular and biological characteristic species from a sample. The introduction of polyatomic ion beams, particularly C60, for TOF-SIMS analysis has created a whole new application of molecular depth profiling and 3D molecular imaging. However, such analyses, particularly at high lateral resolution, are severely hampered by the accompanying mass spectrometry associated with current TOF-SIMS instruments. Hence, we have developed an instrument that overcomes many of the drawbacks of current TOF-SIMS spectrometers by removing the need to pulse the primary ion beam. The instrument samples the secondary ions using a buncher that feeds into a specially designed time-of-flight analyzer. We have validated this new instrumental concept by analyzing a number of biological samples generating 2D and 3D images showing molecular localization on a subcellular scale, over a practical time frame, while maintaining high mass resolution. We also demonstrate large area mapping and the MS/MS capability of the instrument.

Transflection-mode FTIR spectroscopy has become a popular method of measuring spectra from biomedical and other samples due to the relative low cost of substrates compared to transmission windows, and a higher absorbance due to a double pass through the same sample approximately doubling the effective path length. In this publication we state an optical description of samples on multilayer low-e reflective substrates. Using this model we are able to explain in detail the so-called electric-field standing wave effect and rationalise the non-linear change in absorbance with sample thickness. The ramifications of this non-linear change, for imaging and classification systems, where a model is built from tissue sectioned at a particular thickness and compared with tissue of a different thickness are discussed. We show that spectra can be distorted such that classification fails leading to inaccurate tissue segmentation which may have subsequent implications for disease diagnostics applications.

Time-of-flight secondary ion mass spectrometry (ToFSIMS) is being applied increasingly to the study of biological systems where the chemical specificity of mass spectrometry and the high lateral resolution imaging capabilities can be exploited. Here we report a comparison of two cell sample preparation methods and demonstrate how they influence the outcome of the ToFSIMS analysis for three-dimensional (3D) imaging of biological cells using our novel buncher-ToF instrument (J105 3D Chemical Imager) equipped with a C(60) primary ion beam. Cells were analysed fixed and freeze-dried and non-fixed, frozen-hydrated. It is concluded that maintaining the cells in a non-fixed frozen-hydrated state during the analysis helps reduce chemical redistribution, producing cleaner spectra and improved chemical contrast in both 2D and 3D imaging. Insights into data interpretation are included and we present methods for 3D reconstruction of the data using multivariate analysis techniques.