Scanning tunneling microscopy under full electrochemical potential control (in situ STM) has been used extensively as an efficient method to characterize microstructures at solid/liquid interfaces at the atomic and molecular levels. However, under ambient conditions oxygen may interfere in open in situ STM systems by diffusion through the solutions. Such interference can be serious for oxygen sensitive systems both for the target molecules themselves and for chemical linker molecules bound to the electrode surface for example, via Au-S bonding. This is strikingly illustrated in the present report. We present here oxygen-free in situ STM, in which the samples are contained in an argon atmosphere which removes oxygen in the system. Interference from oxygen is profoundly reduced in this setup and demonstrated by three examples. Two of these are in situ STM of the intermediate-size sulfur containing organic molecule cysteamine and the biomolecule homocysteine. The third example is the iron-sulfur protein ferredoxin (Pyrococcus furiosus). Monolayers of these molecules on Au(111) are imaged to molecular resolution and the images compared in the presence and absence of oxygen. In all the cases, the STM image quality of the monolayer structures has improved significantly in the absence of oxygen. Reasons for the drastic effects of dioxygen based on both chemical surface reactivity and electron scattering effects are discussed. These observations are important in general for STM of oxygen sensitive chemical and biological samples. (c) 2006 Elsevier B.V. All rights reserved.