CONFOCAL FLUORESCENCE MICROSCOPIC IMAGING FOR INVESTIGATING THE ANALYTE DISTRIBUTION IN MALDI MATRICES

The analytical performance of matrix-assisted laser desorption/ionizat ion (MALDI) mass spectrometry is strongly influenced by the method of analyte and matrix preparation. We report a nonintrusive method based on laser confocal microscopic imaging technology to examine the MALDI samples prepared by various protocols. In this method, the analyte is tagged with a fluorescent group, The matric and analyte are prepared u nder the same conditions as those used in conventional MALDI experimen ts, It is demonstrated that: confocal microscopy can provide clear, th ree-dimensional images of sample crystals as well as the analyte distr ibution within the crystals. It is shown that the analyte is incorpora ted into the matrix crystals for all the sample preparation protocols examined. Moreover, the confocal microscopic images reveal that, with the use of a dried-droplet method for sample/matrix preparation, the a nalyte is not uniformly distributed within the matrix crystals, In sam e crystals, no analyte is incorporated. In addition, it is found that large crystals formed using a slow growth process display a more unifo rm analyte distribution. Relatively more uniform analyte distribution is observed for samples prepared with the formation of microcrystals. The possible correlation between the ion signal variations observed in MALDI and the uniformity of the analyte distribution obtained by the confocal microscopic imaging method is discussed. Finally, a double-im aging method involving the use of two analytes with different labeling groups is demonstrated. It is found that different analytes are not c oherently distributed in the matrix crystals.