In situ 3D characterization of a filtration cake obtained with a model bidisperse suspension containing two sizes of fluorescent particles (1 mu m and 4.8 mu m) has been performed using Confocal Laser Scanning Microscopy (CLSM). The suspensions were dead-end filtered through 0.8 mu m pore diameters silicon nitride microsieves, under a constant flow rate. The membrane was put in a specific filtration chamber previously designed [1], allowing direct on-line microscopic observations of particles deposition. The final position of the first particles arriving on the membrane was investigated. Then, the online deposition of the following particles was recorded and, thus, the cake build-up could be analyzed layer by layer. In addition, to analyze the protective effect of a layer of large particles, 1 mu m particles were filtrated over a previously deposited cake formed with the 4.8 mu m microspheres. 2014 Elsevier B.V. All rights reserved.