Marco Consumi oral presentation (PB3-Thu1-2-3)
Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS): a promising tool for extracellular matrix ECM analysis
1 Dipartimento di Biotecnologie Chimica e Farmacia, University of Siena, , 53100 Siena, Italy
2 CSGI Center for colloid and surface science, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
The use of metal cluster and polyatomic primary ions greatly enhances the capability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to tackle the analysis of complex biological matrices. Biological materials present a greatest challenge, but also probably the greatest opportunity to apply this label-free analysis.
Time-of-flight secondary ion mass spectrometry was employed to study extracellular polymeric substances from different biological systems, describing how the change of different parameters like pH, temperature etc. will influence the chemical composition of ECM.
The first challenges facing ToF-SIMS in biological studies is the sample preparation, essential when acquiring either meaningful SIMS spectra or images.
In order to prepare ECM for analysis in vacuum, the 3D integrity of the sample must be preserved in the solid state with micrometer precision. We explored few strategies for cell preservation including freeze-drying, freeze-etching, freeze-fracturing, chemical fixation, and sugar vitrification. [2,3] These methods are proved successfully maintaining the physical state of the biological materials. However, when also the chemistry of the sample should be maintained, the situation becomes more complicated and there are few objective studies that seek to assess the usefulness of each preparation protocol. It is clear that fast freezing in liquid propane or isopentane is effective in ensuring that cell or tissue structure is not damaged by water crystallization. Freeze-fracturing of cells or tissues is able to expose regions of interest and it would seem to provide some advantages. The fracturing process is best carried out inside the vacuum system to prevent condensation of water ice on the sample.
Despite freeze-fracturing probably being the best approach, because it is quicker and easier, sample preparation is mostly carried out using freeze-drying approaches. Recently, we developed a special device where the frozen material, cells, bacteria or tissue are placed
between two plates and the whole device is cooled under nitrogen to prevent condensation of water ice on the sample. After cooling, plates are flipped backward to fracture the sample and provide the surfaces of interest ready for analysis