Imaging optical thicknesses and separation distances of phospholipid vesicles at solid surfaces

We present the application of reflection interference contrast
microscopy (RICM) (1) to map the optical density of supported
bilayers and vesicles and (2) to image the contact profile of
phospholipid vesicles at surfaces. The resolution in the surface
profile is 0.2 $\mu$m laterally and 1 nm out of plane. The optical
thickness of the membrane can be determined with 0.2 nm accuracy.
We outline the theoretical basis of RICM and derive the
interference intensities of adhering vesicles from first
principles. An analytical expression for the decaying contrast of
the intrference fringes is given. The contact contour of vesicles
is determined for various substrates. We further demonstrate that
deposition of a magnesium fluoride layer on the glass substrate
enhances the contrast and allows the optical density of adsorbed
membranes to be imaged. By contrast variation of the buffer
solution, the layer thicknesses and the indices of refraction can
be measured. The novel method was applied to image lipid domains of
different chain lengths in a substrate supported monolayer.