The quantification of biofilm structure based on image analysis requires a statistical measure like representative elemental areas (REA) to determine the necessary size of biofilm area to be imaged. In this study, REAs for biofilm structure were calculated for the descriptors Gray level and Correlation (COR) derived from a spatial gray level dependence matrix analysis (SGLDM). An important difference between these two descriptors is their response to structural features at different spatial scales. Gray level is a scale-independent descriptor, whereas COR is scale-dependent. For scale-in dependent descriptors, the size of the individual images is not relevant when determining REAs. This is in contrast to scale-dependent descriptors for which REAs can only be determined when the area of each image covers the range of structural variability of the biofilm. We used COR to analyze scale dependence of structural heterogeneity at different length scales. A characteristic length of 400 mu m in biofilm images provides structural information relevant for mass transport phenomena in biofilms. Overall REAs for gray level and COR were on average 3.4 mm(2). The scale-dependent descriptor COR could not in all cases accurately be determined from combining individual image analysis results-even when the combined area resulted in the REA. Microscope and camera specifications define the upper and lower limit of detectable characteristic length that can be extracted from images and should therefore be considered in the experimental design when choosing an imaging technique.