Photocleavable oligohistidine peptides (POHP) allow in situ spatial organization of multiple His-tagged proteins onto surfaces functionalized with tris(nitrilotriacetic acid) (tris-NTA). Here, a second generation of POHPs is presented with improved photoresponse and site-specifi c covalent coupling is introduced for generating stable protein assemblies. POHPs with different numbers of histidine residues and a photocleavable linker based on the 4,5-dimethoxy-o-nitrophenyl ethyl chromophore are prepared. These peptides show better photosensitivity than the previously used o-nitrophenyl ethyl derivative. Effi cient and stable caging of tris-NTAfunctionalized surfaces by POHPs comprising 12 histidine residues is demonstrated by multiparameter solid-phase detection techniques. Laser lithographic uncaging by photofragmentation of the POHPs is possible with substantially reduced photodamage as compared to previous approaches. Thus, in situ micropatterning of His-tagged proteins under physiological conditions is demonstrated for the fi rst time. In combination with a short peptide tag for a site-specifi c enzymatic coupling reaction, covalent immobilization of multiple proteins into target micropatterns is possible under physiological conditions. ; Rodríguez Muñiz, GM.; Gropeanu, M.; Bhagawati, M.; Gropeanu, RA.; Sundaram, S.; Piehler, J.; Campo, AD. (2013). A Versatile Toolbox for Multiplexed Protein Micropatterning by Laser Lithography. Small. 9(6):838-845. doi:10.1002/smll.201201901. ; Senia ; 838 ; 845 ; 9 ; 6