High spatial resolution and large area thickness mapping of label-free protein microarray has been achieved using imaging ellipsometry (IE) under optimized conditions. The protein patterns with feature size down to 8 x 8 mu m(2) was readily imaged, and the binding between the surface immobilized antigen and the antibody was monitored. Quantitative thickness analysis of antibody-antigen binding on the 32 x 32 mu m(2) micron spots was successfully performed, and we have obtained a limit of detection as low as 1.2 pg/spot. This work demonstrates that appropriately optimized LE could be used as a highly sensitive and high through-put label-free technique for studying surface antigen-antibody recognition in sub-40 mu m scale. (C) 2011 Elsevier Inc. All rights reserved.