Influence of Sample Preparation in Digital Holographic Phase Contrast Microscopy

Quantitative phase contrast imaging with digital holographic microscopy (DHM) enables a label-free analysis of living and fixed cellular specimens. For DHM measurements on fixed adherent cells the sample preparation has to be considered. The embedding medium, e. g. glycerin, affects the integral cellular refractive index and thus leads to an increased uncertainty in the calculation of the cell thickness from the measured phase distributions. Comparative DHM investigations have been carried out for the example of formalin-fixed tumor cells in different embedding media to quantify the influence of the cell preparation on the DHM phase contrast. An empirical model is fitted to the phase data in order to describe the cell curvature and to determine the maximum phase contrast. The obtained results show that it is possible to quantify the influence of the embedding medium by cell specific models and the obtained conversion factors are discussed.