Imaging mass spectrometry technology is rapidly developing. New desorption and ionization methods allow access to an increasingly large number of biomolecular systems. Sensitivity, speed and spatial resolution are continuously improving with new technologies such as cluster ion sources and microscope mode imaging approaches. MALDI imaging and SIMS are providing complementary molecular insights into biomolecular distributions on the surfaces of tissue sections and cells and demonstrate the great promise of biomolecular mass spectrometric imaging. There is one element, all MS imaging researchers agree upon. Sample preparation is crucial to the success of the method. Unfortunately, each application and each MS imaging technology requires a different type of sample preparation. Matrix coating, metal coating, sample morphology, sample history, and local chemical environment all influence the desorption and ionization mechanisms that lie at the basis of all imaging techniques. As images by themselves are a semi-quantitative representation of molecular distributions this obviously raises questions on their reliability. In this paper, a discussion will be devoted to the basic sample preparation requirements for biomedical imaging mass spectrometry. The differences and similarities for the two major imaging MS methods, SIMS and MALDI, will be addressed. Examples of extreme compound suppression as well as different matrix preparation methods for ME-SIMS and MALDI will be discussed. (C) 2008 Elsevier B. V. All rights reserved.