Low Coherence Quantitative Phase Microscopy and Its Clinical Applications

Poola, Praveen Kumar and John, Renu (2018) Low Coherence Quantitative Phase Microscopy and Its Clinical Applications. PhD thesis, Indian institute of technology Hyderabad.

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Abstract

Most of the biological samples such as cells and tissues are phase objects, i.e. absorb little light, resulting in only minor changes in the amplitude of transmitted light through the cell. Since the human eye relies on changes in amplitude of a light wave, cells can be difficult to visualize using a light microscope without a system to enhance cell contrast. One widespread solution is to use dyes that provide molecular specificity. However, involve procedures (fixation) incompatible with live cell imaging. Even labels designed for use with live cell imaging can cause perturbation to normal cellular function and concentrationdependent toxicity effects. Photo-toxicity poses additional barriers to imaging native cell behavior, as the light intensity required to excite a fluorophore can cause cells to behave abnormally or die. Photo-toxicity also adversely affect cell physiology, health, behavior, movement and shape by various mechanisms. Furthermore, label-free techniques enable researchers to avoid the cost of time-intensive dye optimization or stable fluorescentreporter cell line generation. Rather than requiring contrast-enhancing dyes, label-free solutions rely on components of the optical setup that exploit cells inherent contrast mechanisms (thickness and refractive index (RI)) to create image contrast. Abbe theory of image formation phenomenon opened the doors to formulating the problem of contrast precisely like in interferometry. Based on this idea, in the year 1930, Zernike developed phase contrast microscopy (PCM). In the PCM, the contrast is generated by introducing  2 or 3 2 rad into the scattered light from the sample. The PCM technique is a major advancement in the intrinsic contrast imaging, as it reveals inner details of transparent structures without staining or tagging. However, the PCM technique is plagued with significant artifacts such as the halos in the images. In addition to the above PCM technique, there exists other techniques that can also provide the intrinsic contrast from the phase objects, such as dark-field microscopy and Nomarski differential interference contrast microscopy (DIC). The above mentioned techniques are primarily successful in providing a qualitative visualization of phase, however, a quantitative estimation of phase is not feasible.

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