In situ non-invasive 3D characterization of membrane fouling was achieved using femtosecond near infrared non-linear optical imaging together with a novel crossflow filtration module. Washed fluorophore-labelled yeast suspensions were filtered through Millipore 0.22 mu m mixed cellulose ester membranes and the fouling layer was imaged at different times throughout the experiment. Based on the 3D femtosecond images, it has been possible to identify fine structural features of the cake and to measure the thickness of the filter cake formed on the microfiltration (MF) membranes. Our findings reveal that low concentration feeds result in the initial formation of a patchy monolayer of cells leading to a multilayered cake, whilst at higher concentrations a multilayer cake forms rapidly. For patchy cakes, the technique offers greater resolution than that which is achievable with the direct observation through membrane technique. Deposited cell aggregates and broken fragments of cells can clearly be imaged. For thick cakes, it has been possible to image up to depths 45 mu m below the cake surface in the present work. (c) 2006 Elsevier B.V. All rights reserved.