Refractive Index Changes of Cells and Cellular Compartments Upon Paraformaldehyde Fixation Acquired by Tomographic Phase Microscopy

Three-dimensional quantitative phase imaging is an emerging method, which provides the 3D distribution of the refractive index (RI) and the dry mass in live and fixed cells as well as in tissues. However, an insufficiently answered question is the influence of chemical cell fixation procedures on the results of RI reconstructions. Therefore, this work is devoted to systematic investigations on the RI in cellular organelles of live and fixed cells including nucleus, nucleolus, nucleoplasm, and cytoplasm. The research was carried out on four different cell lines using a common paraformaldehyde (PFA)-based fixation protocol. The selected cell types represent the diversity of mammalian cells and therefore the results presented provide a picture of fixation caused RI changes in a broader context. A commercial Tomocube HT-1S device was used for 3D RI acquisition. The changes in the RI values after the fixation process are detected in the reconstructed phase distributions and amount to the order of 10(-3). The RI values decrease and the observed RI changes are found to be different between various cell lines; however, all of them show the most significant loss in the nucleolus. In conclusion, our study demonstrates the evident need for standardized preparation procedures in phase tomographic measurements. (c) 2020 International Society for Advancement of Cytometry

Automated summary

Three-dimensional quantitative phase imaging provides 3D distribution of refractive index and dry mass in cells and tissues. This study investigates the influence of chemical cell fixation on RI in cellular organelles, showing RI changes, especially in the nucleolus, after fixation. Standardized preparation procedures are needed for phase tomographic measurements.