Rationale To discover the degree to which water-containing cluster beams increase secondary ion yield and reduce the matrix effect in time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging of biological tissue. Methods The positive SIMS ion yields from model compounds, mouse brain lipid extract and mouse brain tissue together with mouse brain images were compared using 20 keV C60+, Ar2000+, water-doped Ar2000+ and pure (H2O)6000+ primary beams. Results Water-containing cluster beams where the beam energy per nucleon (E/nucleon) ≈ 0.2 eV are optimum for enhancing ion yields dependent on protonation. Ion yield enhancements over those observed using Ar2000+ lie in the range 10 to >100 using the (H2O)6000+ beam, while with water-doped (H2O)Ar2000+ they lie in the 4 to 10 range. The two water-containing beams appear to be optimum for tissue imaging and show strong evidence of increasing yields from molecules that experience matrix suppression under other primary beams. Conclusions The application of water-containing primary beams is suggested for biological SIMS imaging applications, particularly if the beam energy can be raised to 40 keV or higher to further increase ion yield and enhance spatial resolution to ≤1 µm. © 2015 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.