C.elegans is a biomedical key model organism as it is a simple, easy to maintain eukaryote, which shares many gene homologues with higher mammals. As each of its cells has been traced during development and characterized by light microscopy, mass spectrometry-based time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging seems to promise exciting new insights. In this study, we have investigated simple but basic factors for ToF-SIMS-based imaging of C.elegans. By comparing chemical standards and two authentic C. elegans mutant extracts (N2 and Daf-2), we found that for our purposes, Bi3+ is better suited due to its higher mass and spatial resolution. We also investigated the use of light microscopy slides as imaging substrates and gold coating as standard for mass calibration in higher mass ranges. Our findings and preliminary imaging results show that ToF-SIMS is a well-suited platform for mass spectrometry imaging. Copyright © 2012 John Wiley & Sons, Ltd.