Holotomographic investigation of an influence of PFA cell fixation process on refractive index of cellular organelles in epithelial cells

Abstract

Holographic Tomography (HT) is currently the most common tool in biomedical applications, which provides 3D distribution of refractive index (RI) and dry mass (dm) in biological cells and tissues. It uses the refractive index (RI) as contrast agent for a single cell or tissue analysis, which is now considered as important biophysical feature to be applied for future applications in digital phase histopathology or liquid biopsy, e.g., supported by deep learning procedures. RI investigations at cellular level may be performed based on living or chemically fixed cells. In this work we had focused on investigations of the influence of PFA fixation process on RI in cellular organelles: nucleus, nucleolus, nucleoplasm and cytoplasm. The research was carried out on the NRK-52E rat kidney epithelial cell line. Epithelial cells were chosen for the experiment due to the fact that they belong to the basic building cells of the human body and are often used for normal and extended toxicity experiments with nano particles. A commercial Tomocube HT-1S device was used for the research. RI of live and fixed cells was measured. Changes in RI after fixation are observed at the order of 10-3. The RI values decrease, and the mass density is found reduced in the fixated cells. In conclusion, our results demonstrate the need of standard procedures for the preparation of biological samples for phase tomographic measurements in the case of chemically fixed cells. Moreover the requirement for conversion factors to retrieve accurate RI and dm values that are comparable to living cells is discussed.