Membrane biofouling in membrane bioreactor (MBR) was analyzed quantitatively using a quantitative image analysis technique combined with confocal laser scanning microscopy (CLSM). The CLSM-Image analysis technique was (i) used to quantify the structure of bio-cake relating to the spatial change in the architecture, (ii) then applied to calculate the contribution of bacterial cell and extra-cellular polymeric substances (EPSs) to bio-cake resistances (R-c). A variation in the structural parameters of the bio-cake was found for different fluxes. EPS was more prevalent for the system operated under the lowest flux of 13 L/(m(2) h). At this "subcritical'' flux, the amount of EPS in the final bio-cake was greater than the amount of bacterial cells. This flux also gave the greatest rate of resistance rise towards the end of the period of operation. The profiles of each component (cell and EPSs) with in the bio-cake were used to estimate the cake resistance (R-c). A particle size of less than 1 mu m was required to have correspondence between theory and measurement. It was deduced that the steep TMP rise (labeled by others as Stage 3) is due to a combination of two mechanisms: inhomogeneous pore loss and changes in percolation due to EPS accumulation. (c) 2012 Elsevier B.V. All rights reserved.