Surface plasmon resonance microscopy: achieving a quantitative optical response
Alexander W. Peterson, Michael W. Halter, Anne L. Plant, John T. Elliott
Surface plasmon resonance imaging (SPRI) allows real-time label-free imaging based on index of refraction, and changes in index of refraction at an interface. SPRI can be carried out on a microscope by launching light into a sample, and collecting reflected light through a high numerical aperture microscope objective. The SPR microscope enables spatial resolution that approaches the diffraction limit, and has a dynamic range that allows detection of subnanometer to submicrometer changes in thickness of biological material at a surface. However, unambiguous quantitative interpretation of SPR changes using the microscope system could not be achieved using the Fresnel model because of polarization dependent attenuation and optical aberration that occurs in the high numerical aperture objective. To overcome this problem, we applied an optical model to correct for polarization diattenuation and optical aberrations in the SPRI data, and developed a procedure to calibrate reflectivity to index of refraction values. The calibration and correction strategy was validated by comparing the known indices of refraction of bulk materials with SPRI data interpreted with the corrected Fresnel model. Subsequently, we used SPR microscopy to evaluate the index of refraction for a series of polymer microspheres in aqueous media and validated the quality of the measurement with quantitative phase microscopy.