New modeling of reflection interference contrast microscopy including polarization and numerical aperture effects: application to nanometric distance measurements and object profile reconstruction.

摘要: We have developed a new and improved optical model of reflection interference contrast microscopy (RICM) to determine with precision few nanometers the absolute thickness h thin films on flat surface in immersed conditions. The takes into account multiple reflections between planar multistratified object, finite aperture illumination (INA), and, for first time, polarization light. RICM intensity I is typically oscillating h. introduce normalization procedure that uses extrema same oscillation order both experimental theoretical values permits us avoid significant error height determination, especially at high INA. also show how problem solution degeneracy can be solved by taking pictures two different INA values. applied filled polystyrene beads giant unilamellar vesicles radius 10-40 microm sitting glass substrate. profiles I(h) fitted up three orders, positions are correct five seven orders. distance shape objects near substrate about 5 nm range from 0 500 nm, even under large numerical method valuable dynamic experiments living cells where apertures required.