Reliably measuring the physicochemical properties of protein thin layers deposited on surfaces is critical to understanding the surface chemistry, biocompatibility, and performance of implanted biomaterials. Here we apply a series of multi-fluorophore labeled Bovine Serum Albumin (BSA) proteins as model probes to investigate surface-induced conformational changes of BSA by the use of a confocal Fluorescence Lifetime Imaging Microscopy and Forster Resonance Energy Transfer (FLIM-FRET) method. In this FLIM-FRET approach we study six different constructs where the BSA is covalently linked to one (BSA-F1) or five (BSA-F5) fluorescein molecules, one (BSA-T1) or seven (BSA-T7) rhodamine molecules, and hetero labeled with both (BSA-F4-T2 and BSA-F6-T1). The fluorescence intensity and decays were simultaneously measured at two different emission regions (green and red channels) of the labeled BSA deposited on substrates of different hydrophilicity and hydrophobicity. To generate reliable data, several different regions (10(4) mu m(2) in each case) of the surfaces were scanned for each measurement. The amplitude-weighted lifetimes, obtained from the fluorescence decay parameters, are discussed based on the average distance between the conjugated fluorophores acting as a donor and acceptor pair in the Energy Transfer framework. The number of probes conjugated has significant effects on the fluorescence emission intensity and lifetimes in solution and on surfaces. The BSA-F4-T2 constructs showed a significant ability to differentiate using lifetime the hydrophilicity and hydrophobicity of the surfaces, by detecting local expansion and contraction of protein structure in the deposited layers. Using these multiple labeled BSA probes in conjunction with FLIM-FRET can provide a way to assess structural changes in proteins induced by variations in surface chemistry of biomaterials. (C) 2010 Elsevier B.V. All rights reserved.