RATIONALE Microscope mode imaging for secondary ion mass spectrometry is a technique with the promise of simultaneous high spatial resolution and high‐speed imaging of biomolecules from complex surfaces. Technological developments such as new position‐sensitive detectors, in combination with polyatomic primary ion sources, are required to exploit the full potential of microscope mode mass spectrometry imaging, i.e. to efficiently push the limits of ultra‐high spatial resolution, sample throughput and sensitivity. METHODS In this work, a C 60 primary source was combined with a commercial mass microscope for microscope mode secondary ion mass spectrometry imaging. The detector setup is a pixelated detector from the Medipix/Timepix family with high‐voltage post‐acceleration capabilities. The system's mass spectral and imaging performance is tested with various benchmark samples and thin tissue sections. RESULTS The high secondary ion yield (with respect to 'traditional' monatomic primary ion sources) of the C 60 primary ion source and the increased sensitivity of the high voltage detector setup improve microscope mode secondary ion mass spectrometry imaging. The analysis time and the signal‐to‐noise ratio are improved compared with other microscope mode imaging systems, all at high spatial resolution. CONCLUSIONS We have demonstrated the unique capabilities of a C 60 ion microscope with a Timepix detector for high spatial resolution microscope mode secondary ion mass spectrometry imaging. Copyright © 2013 John Wiley & Sons, Ltd.