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The effects of chemical fixation on the cellular nanostructure.

ABSTRACT: 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.

SUBMITTER: Li Y

PROVIDER: S-EPMC5726765 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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The effects of chemical fixation on the cellular nanostructure.

Li Yue Y Almassalha Luay M LM Chandler John E JE Zhou Xiang X Stypula-Cyrus Yolanda E YE Hujsak Karl A KA Roth Eric W EW Bleher Reiner R Subramanian Hariharan H Szleifer Igal I Dravid Vinayak P VP Backman Vadim V

Experimental cell research 20170630 2

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 fi ...[more]

PMID: 28673821

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Project description:Chemical cytometry is a powerful tool for measuring biological processes such as enzymatic signaling at the single cell level. Among these technologies, single-cell capillary zone electrophoresis (CZE) has emerged as a powerful tool to assay a wide range of cellular metabolites. However, analysis of dynamic processes within cells remains challenging as signaling pathways are rapidly altered in response to changes in the cellular environment, including cell manipulation and storage. To address these limitations, we describe a method for chemical fixation of cells to stop the cellular reactions to preserve the integrity of key signaling molecules or reporters within the cell and to enable the cell to act as a storage reservoir for the reporter and its metabolites prior to assay by single-cell CZE. Fluorescent phosphatidylinositol 4,5-bisphosphate reporters were loaded into cells and the cells were chemically fixed and stored prior to analysis. The reporter and its metabolites were electrophoretically separated by single-cell CZE. Chemical fixation parameters such as fixative, fixation time, storage solution, storage duration, and extraction solution were optimized. When cells were loaded with a fluorescent C6- or C16-PIP2 followed by glutaraldehyde fixation and immediate analysis, 24±2% and 139±12% of the lipid was recoverable, respectively, when compared to an unfixed control. Storage of the cells for 24h yielded recoverable lipid of 61±3% (C6-PIP2) and 55±5% (C16-PIP2) when compared to cells analyzed immediately after fixation. The metabolites observed with and without fixation were identical. Measurement of phospholipase C activity in single leukemic cells in response to an agonist demonstrated the capability of chemical fixation coupled to single-cell CZE to yield an accurate snapshot of cellular reactions with the probe. This methodology enables cell assay with the reporter to be separated in space and time from reporter metabolite quantification while preserving assay integrity.

Project description:It has been reported that supply of nitrate to culture solution rapidly and reversibly inhibits nodule growth and nitrogen fixation activity of soybean. In this study, the effects of ammonium, urea, or glutamine on nodule growth and nitrogen fixation activity are compared with that for nitrate. Soybean plants were cultivated with a nitrogen-free nutrient solution, then 1 mM-N of nitrate, ammonium, glutamine, or urea were supplied from 12 DAP until 17 DAP. Repression of nodule growth and nitrogen fixation activity at 17 DAP were observed by ammonium, urea, and glutamine like nitrate, although the inhibitory effects were milder than nitrate. The removal of nitrogen from the culture solutions after nitrogen treatments resulted in a recovery of the nodule growth. It was found that the glutamine treatment followed by N-free cultivation gave highest nitrogen fixation activity about two times of the control. Tracer experiments with 15N and 13C were performed to evaluate the translocation of N and C to the different tissues. Culture solutions containing a 15N-labeled nitrogen source were supplied from 21 DAP, and the whole shoots were exposed to 13CO2 for 60 min on 23 DAP, and plants were harvested on 24 DAP. The percentage distribution of 15N in nodules was highest for ammonium (1.4%) followed by glutamine (0.78%), urea (0.32%) and nitrate (0.25%). The percentage distribution of 13C in the nodules was highest for the control (11.5%) followed by urea (5.8%), glutamine (2.6%), ammonium (2.3%), and nitrate (2.3%). The inhibitory effects of nitrogen compounds appeared to be related to a decrease in photoassimilate partitioning in the nodules, rather than 15N transport into the nodules. The free amino acid concentrations after nitrogen treatments were increased in the nodules and leaves by nitrate, in the roots by ammonium, in the stems by urea, and the roots, stems, and leaves by glutamine treatment. The concentrations of asparagine, aspartate, and glutamine were increased after nitrogen treatments. By the long-term supply of nitrogen for 2-weeks, nitrate significantly increased the lateral roots and leaf growth. The long-term supply of urea and glutamine also promoted the lateral roots and leaf growth, but ammonium suppressed them.

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The effects of chemical fixation on the cellular nanostructure.

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The effects of chemical fixation on the cellular nanostructure.

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