The study of molecular signaling processes related to disease requires not only the detection and analysis of the molecules involved but also the evaluation of their spatial organization. Many molecular imaging modalities have addressed the different levels of complexity of cancer, and mass spectrometry imaging was added to the multimodal toolbox three decades ago. We have developed a multimodal approach that combines a number of innovative mass spectrometric imaging approaches with optical imaging, magnetic resonance imaging and targeted immunohistochemistry images to refine our understanding of disease progression. In addition we have integrated state-of-the-art quantitative proteomics and metabolomic studies of selected tissues in our molecular imaging workflow. We will discuss applications of new chemical microscopes in biomedical tissue analysis targeting cancer research in particular, and showhow the multimodal mass spectrometry imaging can elucidate the way in which local environments can influence signaling pathways in diseases..