Nano- and micrometer site-specific chemical surface patterning has become important to the field of biology and medicine.Precise spatial control of chemistry on surfaces provides an essential platform for the directed attachment of bioactive molecules, relevant for numerous applications such as biosensors, tissue engineering and fundamental studies of molecular and cell and micro-biology. New patterning methods based on colloidal crystals will be presented, in which colloids of one size or binary colloids are generated on surfaces over large areas. The crystal layers are used as masks against deposition of plasma polymers and/or metals such as gold to create complex patterns of dimensions ranging from mms to sub 100nms and are useful for post-modification with different chemistries for site-specific immobilisation of biomolecules. This includes using chemistries for preventing non-specific adsorption of proteins and attachment of cells, including new ways of generating high graft density polymer brushes. The presentation will also demonstrate the importance of using surface sensitive analytical tools to prove the presence of the different surface chemistries. These include x-ray photoelectron spectroscopy (XPS), and high resolution time-of-flight secondary mass spectrometry (ToF-SIMS) imaging.Finally, the presentation will provide details of preliminary cell attachment results onto colloidal crystals where methods for stabilizing the crystals are being developed.