Abstract
In this study, we aimed at establishing a procedure for normalizing mass spectra to improve ion distribution images, i.e., sharply defined tissue edges and increased dynamic range, acquired by mass spectrometric imaging (MSI). A crucial problem in MSI is that variations in ionization efficiencies are observed among spectra, which arises from the heterogeneous matrix-analyte crystallization and sublimation during measurement. Such variation could generate incorrect analyte distribution images. For solving this problem, we performed spectrum normalization using an external standard (ES) compound spiked in the matrix solution. We selected methylcarbamyl platelet-activating factor (C-PAF) (C-16) as the ES compound by considering the following two criteria: (1) no other mass peaks overlap the peak of the ES compound, and (2) the ES compound has sufficient ionization capability on the tissue section, in which numerous biological compounds compete to ionize. For evaluating the optimal concentration of C-PAF, we analyzed ion images of coronal mouse brain sections by MSI using matrix solutions containing different concentrations of C-PAF (0, 0.5, 5, 25, and 50 μg/mL). As a result, we found that the optimal concentration of C-PAF for normalization is 50 μg/mL. Employing the procedure, we show improved ion images of phosphatidylcholine (PC) molecular species in the mouse brain section.
