The immobilization of trypsin on porous glycidyl methacrylate (GMA-GDMA) beads has been investigated. In particular, the distribution within the beads of trypsin and of dextran used for hydrophilizing the bead surface prior to protein immobilization was investigated with confocal microscopy. For the system investigated, the fluorescence intensity profiles obtained when using borate buffer as an ambient solution displayed a distinct minimum at the center of the beads, irrespective of the observation depth. However, by reduction of the refractive index difference between the solution and the beads through the addition of glucose to the aqueous solution, artifacts relating to optical length differences could be reduced. For both low molecular weight fluorescein isothiocyanate (FITC), FITC-labeled trypsin, and FITC-labeled dextran, an essentially homogeneous distribution throughout the beads was observed. This simple "contrast matching" method seems therefore to be an interesting tool when investigating the distribution of immobilized protein in porous chromatography media. Copyright 1999 Academic Press.