Results
To keep this chapter as clear as possible, we divided it into three subchapters: tissue washing (1), matrix spraying nozzle height over the sample, and the number of matrix layers in positive (2) and negative (3) ionization modes. DHB, norharmane, and DAN matrices were tested in the positive ionization mode. In the negative ionization mode, norharmane, NEDC, and 9AA matrices were used. Below there is a brief description of the optimization procedures.
Tissue washing
Some Authors recommend applying the tissue washing procedure before analysis to remove substances interfering with the ionization process19,21. The procedure is easy: the sample on the ITO glass should be immersed into a solvent a few times and dried. To test this approach, we used the procedure described by Angel et al.19: two slices were washed with 50 mM ammonium acetate by immersing them for 10 s and subsequent drying in the desiccator for the next 15 minutes.
After washing, we observed four types of effects influencing the final results:
(1) some substances were completely washed away: their ions, typically present in non-washed tissue, were not observed,
(2) the final picture was significantly blurred (see Fig. 2: the difference between both gray and white matters has vanished),
(3) all peak intensities were elevated (probably due to the removal of substances responsible for the ion suppression effect). However, spectra enhancement did not significantly influence the interpretation of the results.
(4) The picture for specific m/z values remains the same before and after washing.
Summing up, washing procedures, if not thoroughly tested to visualize the peaks of interest, usually could not lead to the desired results. It is also important to keep in mind the possibility of delocalization of the molecules and increase the sample variability generated by the washing procedure. So, we decided to perform the measurements in the conditions as unchanged as possible.