We have improved methods reported earlier [11 for sample preparation, imaging and quantifying components in supported lipid bilayers using high-resolution secondary ion mass spectrometry performed with the NanoSIMS 50. By selectively incorporating a unique stable isotope into each component of interest, a component-specific image is generated from the location and intensity of the unique secondary ion signals exclusively produced by each molecule. Up to five species can be simultaneously analyzed. Homogeneous supported lipid bilayers that systematically varied in their isotopic enrichment levels were freeze-dried and analyzed with the NanoSIMS 50. The molecule-specific secondary ion signal intensities had an excellent linear correlation to the isotopically labeled lipid content. Statistically indistinguishable calibration curves were obtained using different sample sets analyzed months apart. Fluid bilayers can be patterned using lithographic methods and the composition of each corralled region varied systematically by simple microfluidic methods. The resulting composition variations can be imaged and quantified. This approach opens the possibility of imaging and quantifying the composition of microdomains within membranes, including protein components, without using bulky labels and with very high lateral resolution and sensitivity. (c) 2006 Elsevier B.V. All rights reserved.