White Light Diffraction Phase Microscopy

Quantitative phase imaging (QPI) techniques are very advantageous compared to the qualitative phase imaging techniques like bright field microscopy, phase contrast microscopy, differential interference contrast microscopy due to measurement of phase information quantitatively. In QPI techniques, there is no need of exogenous contrast agents to stain or tag the specimen. White light diffraction phase microscopy (wDPM) is one of the QPI techniques which is used to quantify the phase information from the samples. The phase information retrieved is dependent on the local refractive index and thickness of the biological cells. So, by measuring the phase one can find the refractive index and/or thickness of the specimen in nanometer scales. In this technique, the acquisition speed is only limited by the sensing device due to its single shot feature, it is better compared to other quantitative phase imaging techniques as its common path geometry provides better temporal phase sensitivity by cancelling out the most mechanisms responsible for noise and with the use of white light source the images are speckle-free. So we achieve better spatial phase sensitivity compared to other techniques. This method has attractive applications in biomedicine. We have developed wDPM set up for extracting phase information from the live yeast cells and sperm cells. The phase is reconstructed from the recorded interferograms by using a fast phase reconstruction algorithm. This algorithm avoids the unwrapping step which is needed in conventional phase reconstruction by Hilbert’s transform method. We have calculated the thickness of the yeast cells and sperm cells and demonstrate a video for visualizing the dynamics of yeast cells.