In the present study, SF5+ and C-60(+) were used as primary ions for sputtering and Bi-3(+) was used as primary ions for analysis. The depth profiling procedure was utilized to make 3D images of the chemistry of single cultured cells and tissue samples of intact intestinal epithelium. The results show sputtering of organic material from cells and tissue with both SF5+ and C-60(+) sources. Cholesterol fragments were found in the superficial layers when sputtering with C-60(+). Spectra were collected revealing the change in yield along the z-axis of the sample. 3D images of the localization of Na, K, phosphocholine and cholesterol were constructed with both ion sources for single cell cultures and the mouse intestine. Cryostate sections of mouse intestine were analysed in 2D and the results were compared with the 3D image of the intestine. The localization of cholesterol and phosphocholine was found to be similar in cryostate sections analysed in two dimensions and the sputtered, freeze-dried intestine analysed in 3D. The comparison of 2D and 3D images suggest that the phosphocholine signal faded with C-60(+) sputtering. In conclusion, both C-60(+) and SF5+ can be used as primary ion sources for sputtering of organic material from cells and tissues. Consecutive analysis with a Bi-3(+) source can be used to obtain image stacks that could be used for reconstruction of 3D images. (C) 2008 Elsevier B. V. All rights reserved.