Research Highlight: “The effects of chemical fixation on the cellular nanostructure”
Yue Li, Luay M. Almassalha, John E. Chandler, Xiang Zhou, Yolanda E. Stypula-Cyrus, Karl A. Hujsak, Eric W. Roth, Reiner Bleher, Hariharan Subramanian, Igal Szleifer, Prof. Vinayak P. Dravid, and Vadim Backman.
Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods.
Full article can be found here.
PWS measurement of Hela Cells at different stages of fixation. The higher value (redder) indicates higher level of mass-density heterogeneity. (A) (C) (E) show live Hela cells without any treatment. (B) After 4% PFA fixation. (D) After 95% EtOH fixation. (F)–(H), after 2.5% GA and 2% FA, after 1% OsO4, and after serial ethanol dehydration. Color scale: (A) (B) (E) (F) from 0.005 to 0.15, (D) from 0.005 to 0.5, (G) and (H) from 0.05 to 0.25. The inset squares are higher magnification images of nucleus and cytoplasm region.