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

Abstract

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.

Bacteria Single-Cell and Photosensitizer Interaction Revealed by Quantitative Phase Imaging

“…However, the RI values of the subcellular structures such as cytoplasm and nucleoid [ 50 ] are changing depending on the kind of medium used [ 32 ]. Additionally, the already reported results of using optical diffraction tomography to reconstruct the 3D-RI distributions of the microalgae [ 24 ], eukaryotic cells [ 52 ], and their internal structures [ 32 ]—chromosomes, cytosol, cell membrane—confirm our concerns because, depending on the type of nutrient medium, osmolality, temperature, or fixation procedures [ 53 ], significant variation of the RI values was observed. Therefore, the use of the DHT or quantitative phase imaging for characterization of single bacteria cells requires standardization and strict control of the cell’s culturing and measurement conditions to obtain comparable and repeatable results.…”

“…on the type of nutrient medium, osmolality, temperature, or fixation procedures [53], significant variation of the RI values was observed. Therefore, the use of the DHT or quantitative phase imaging for characterization of single bacteria cells requires standardization and strict control of the cell's culturing and measurement conditions to obtain comparable and repeatable results.…”

Label-Free Quantitative Phase Imaging Reveals Spatial Heterogeneity of Extracellular Vesicles in Select Colon Disorders

“…A preliminary study concerning the feasibility of differentiating the samples with UC, CRC and surgically removed healthy margins representing normal colonic mucosa based on EV RI values was performed. However, as previously reported, 36 the reconstructed RI-values were affected by the fixation methods, 36 which suggests that the sample preparation procedures can significantly affect the RI data. Therefore, DHT examination was performed on samples prepared with different isolation techniques, including the use of commercially available kits (ExoQuick  TEIK reagents), ultracentrifugation (U) and differential ultracentrifugation on a sucrose cushion (DU).…”

Label-free monitoring of 3D cortical neuronal growth in vitro using optical diffraction tomography

“…However, the RI values in the background were slightly elevated, and some of the original high-RI components in the neurites were eliminated during fixation. A similar effect of the fixation procedure on the RI of cells has been previously reported [57,58]. Although the effect was highly dependent on the cell type, the RI values and contrast between the subcellular components decreased.…”