A novel atmospheric pressure imaging mass spectrometry approach that offers improved lateral resolution (10 μm) using near-infrared femtosecond laser pulses for non-resonant desorption and ionization of sample constituents without the need of a laser-absorbing matrix is demonstrated. As a proof of concept the method was used to image a two-chemical pattern in paper. To demonstrate the ability of the approach to analyze biological tissue, a monolayer of onion epidermis was imaged allowing the chemical visualization of individual cells using mass spectrometry at ambient conditions for the first time. As the spatial resolution is currently limited by the limit of detection of the setup (∼500 fmol limit of detection for citric acid), improvements in sensitivity will increase the achievable spatial resolution. © 2010 American Chemical Society.