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

Abstract

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 100 × / 1.3 NA objective lens, which was not specially selected for minimum wavefront and polarization aberrations, provides OPL noise level of ? 0.5 ?? nm and lateral resolution if ? 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.

Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy

“…, 2011 ). However, OI-DIC provides better image quality and superior resolution (Supplemental Figure S7) because it employs the full numerical apertures of the condenser and the objective lenses, as well as optical subtraction of the images, followed by computation subtraction ( Shribak et al. , 2017 ).…”

Gradient light interference microscopy for 3D imaging of unlabeled specimens

“…DIC with two orthogonal shear directions has been used to obtain 2D quantitative phase images 21 , 22 , whereas Mehta et al 23 reported a partially coherent model for DIC 2D imaging. Shribak et al 24 used liquid crystal modulators to change the polarization directions and phase shift modulation. This setup allows them to record the 2D phase-gradient information at two orthogonal directions and reconstruct the optical phase front.…”

An orientation‐independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model

“…The contrast of DIC images depends on the orientation of specimen and the bias between interfering beams (Oldenbourg & Shribak, 2009). To overcome the limitations of DIC technique, a quantitative OI-DIC microscope has been developed (Shribak & Inoué, 2006;Shribak, 2013;Shribak et al, 2017). OI-DIC provides a novel approach to improving the resolution and the contrast of our images.…”

“…The OPD equals to the product of thickness and refractive index difference and represents the cell dry mass (Barer, 1952;Zangle & Teitell, 2014). The obtained OPD images can be enhanced by using deconvolution or the inverse Riesz transform (Larkin & Fletcher, 2014;Shribak et al, 2017).…”

“…Then we use the 2D Fourier integration of obtained OPD gradient vector map to compute a map of the OPD φ(x,y). (Shribak et al, 2017). For this purpose the OPD gradient vector G (x,y) can be presented as a complex number:…”