Quantitative DIC Microscopy
While fuorescence has become the dominant mode in biological microscopy nowadays for its high molecular contrast, the capability of other modes based on scattering, polarization, and interference, and especially the quantitative aspects of these modes, are more and more frequently overlooked. In particular, most biologists are not familar with the fact that several physical properties of biological samples, such as thickness variation, refractive index distribution and even molecular orientation, can be quantitatively and accurately measured through these modes without introducing fluorophores to the sample of interest. ​
Our quantitative approach in differential interference contrast microscopy is an example that explores the forgotten power of . The issue with traditional Nomarski DIC is that the intensity profile across a given object is nonlinearly related to its differential phase. This means a simple integration will not yield an accurate representation of the object's true phase. In order to solve this problem and linearize the image intensity, a phase shifting DIC approach has been developed. It utilizes a de Senarmont compensator to capture 4 phase shifted images in a given shear direction at 0°, 90°, 180°, and 270°. The process is then repeated in the orthogonal shear direction resulting in 8 total images.​
These phase shifted images can then be combined, using phase retrieval equations intially developed for optical metrology, to create an image where intensity linearly maps to the phase differential in a given direction of shear. Phase integration algorithms can then be used to combine the two phase differential images into a single optical path distance map of the object.​
Recent work on quantitative DIC has been geared towards the automation of the phase shifting process. This will allow for video-rate acquistion of quantitative phase images of live-cell biological processes. Through our collaboration with Boulder Nonlinear Systems we have developed a set of liquid crystal prisms that are capable of variable shear and a liquid crystal phase bias cell that is capable of quickly shifting the phase. These new liquid crystal devices allow for fast acquisition of the images necessary to compute the optical path of a given specimen with the need of a de Senarmont compensator.
