Phase contrast microscopy is a microscopy technique used to enhance contrast of transparent biological samples, by translating phase shift variations of light, as it passes through the sample, into changes of its amplitude, which can be detected via intensity variations. It is a label-free, non-destructive method for observing biological samples, making it widely used. A modern approach to phase contrast microscopy is called Differential phase contrast (DPC) that uses at least one pair of complimentary illumination patterns to reconstruct the final phase image, however, it only qualitatively visualises the phase shift variations. Quantitative phase imaging (QPI) is a microscopy technique that, quantitatively reconstructs phase images using phase variation information, from light passing through a transparent object. In this master’s thesis I used the quantitative differential phase contrast (qDPC) method to quantitatively reconstruct phase shift using the illumination scheme proposed in DPC and the weak object approximation (WOA). I used the Tikhonov regularisation to calculate the phase, for which a regularisation term is added to the least square minimisation, where a regularisation parameter is needed. I determined the optimal parameter using the discrepancy principle, which uses the noise estimation of the images to find the optimal regularisation parameter. Comparing the reconstructed phase images with fluorescence images showed that cell features, such as lipid droplets and cell nuclei, can be visualised with both techniques. Using the qDPC method to reconstruct samples where different types of nanoparticles were mixed with cells showed that nanoparticle aggregates can also be reconstructed on the phase image, however, their calculated phase shift values cannot be taken as accurate due to them introducing a high optical gradient, meaning they violate the WOA. Around nanoparticle aggregates are also present halo artifacts that are a consequence of a big change in refractive index, and influence the quality and accuracy of phase reconstruction. The qDPC method is able to quantitatively reconstruct the phase shift of thin, transparent samples and can be used to visualise certain cell features without the use of toxic cell dyes, which are needed in fluorescence microscopy.
Luka Šalamun (Thu,) studied this question.