Quantitative fluorescence microscopy using supported lipid bilayer standards

Routine quantitative analysis of biomolecule surface density by fluorescence microscopy has been limited by the difficulty of preparing appropriate calibration standards that relate measured fluorescence intensity to actual surface concentration. Supported lipid bilayers are planar fluid films of uniform density and composition which can incorporate a variety of lipidated fluorophores and work well as fluorescence standards. Here, we outline a straightforward strategy to calibrate digital micrographs of fluorescent surfaces such as planar cellular junctions for comparison to supported bilayer standards. It can be implemented with standard microscopy equipment. To illustrate the advantages of this approach, we quantify cell- and bilayer-side protein density patterns in a hybrid immunological synapse between a T-cell and a supported bilayer.