Mapping optical path length and image enhancement using quantitative orientation-independent differential interference contrast microscopy

We describe the principles of using orientation-independent differential interference contrast (OI-DIC) microscopy for mapping optical path length (OPL). Computation of the scalar two-dimensional OPL map is based on an experimentally received map of the OPL gradient vector field. Two methods of contrast enhancement for the OPL image, which reveal hardly visible structures and organelles, are presented. The results obtained can be used for reconstruction of a volume image. We have confirmed that a standard research grade light microscope equipped with the OI-DIC and 100x/1.3 NA objective lens, which was not specially selected for minimum wavefront and polarization aberrations, provides OPL noise level of similar to 0.5 nm and lateral resolution if similar to 300 nm at a wavelength of 546 nm. The new technology is the next step in the development of the DIC microscopy. It can replace standard DIC prisms on existing commercial microscope systems without modification. This will allow biological researchers that already have microscopy setups to expand the performance of their systems. (C) 2017 Society of Photo-Optical Instrumentation Engineers (SPIE)