In Situ Stepwise Surface Analysis of Micropatterned Glass Substrates in Liquids Using Functional Near-Field Scanning Optical Microscopy

Imaging of two adsorbed proteins on micropatterned glass substrates in air and liquid was conducted using atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM). The micropatterned glass substrates were prepared in five steps and evaluated by NSOM at each processing step. The micropatterned samples consisted of alternating 10 μm wide laminin stripes and 20 μm wide bovine serum albumin stripes. The samples containing both protein stripes were imaged “dry” (i.e., in air) using conventional contact AFM, then imaged dry with NSOM, and finally imaged in the hydrated form (i.e., in liquid) using NSOM. When samples were imaged in air, similar topographical features were observed using both AFM and NSOM. However, after the sample was rehydrated and imaged with NSOM in solution, the topography changed significantly. Having confirmed that the “real” surface topography was obtained while the proteins were in the hydrated form, in situ NSOM was used to detect changes in surface topography with nanometer resolution as a function of the micropatterning process. Although lithographic micropatterning provides chemical cues, in situ NSOM demonstrates that topographical cues are also an important aspect of the surface that serves to regulate cellular attachment and migration.