AbstractAnimal cell culture is widely used in biotechnology for the production of many biological products. In situ microscopes acquire images directly from cell suspensions and analyze the images in matters of cell concentration, cell size distribution and cell morphology. Their applicability was already proven for yeast and suspended mammalian cell cultivations. In this work the in situ microscope was utilized to measure the level of colonization of fibroblasts on microcarrier surfaces during cultivation. For this study the murine cell line NIH‐3T3 was used in combination with Cytodex 1 microcarriers. Cultivations were carried out in a 5 L stirred tank bioreactor equipped with the in situ microscope. Images were obtained sequentially with the in situ microscope over the whole cultivation time (900 images per sequence, 7.5 h per sequence on average). For the microcarrier analysis an image analysis algorithm based on a neural network was developed and implemented in the microscope analysis software. Biotechnol. Bioeng. 2008;99: 136–145. © 2007 Wiley Periodicals, Inc.