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Changes in Biomolecular Profile in a Single Nucleolus during Cell Fixation

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    Changes in Biomolecular Profile in a Single Nucleolus during Cell Fixation
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    Institute for Lasers, Photonics and Biophotonics, Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York 14260-3000, United States
    *Phone: 716-645-4148. E-mail: [email protected]
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    Analytical Chemistry

    Cite this: Anal. Chem. 2014, 86, 21, 10909–10916
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    https://doi.org/10.1021/ac503172b
    Published September 29, 2014
    Copyright © 2014 American Chemical Society
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    Abstract

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    Fixation of biological sample is an essential technique applied in order to “freeze” in time the intracellular molecular content. However, fixation induces changes of the cellular molecular structure, which mask physiological distribution of biomolecules and bias interpretation of results. Accurate, sensitive, and comprehensive characterization of changes in biomolecular composition, occurring during fixation, is crucial for proper analysis of experimental data. Here we apply biomolecular component analysis for Raman spectra measured in the same nucleoli of HeLa cells before and after fixation by either formaldehyde solution or by chilled ethanol. It is found that fixation in formaldehyde does not strongly affect the Raman spectra of nucleolar biomolecular components, but may significantly decrease the nucleolar RNA concentration. At the same time, ethanol fixation leads to a proportional increase (up to 40%) in concentrations of nucleolar proteins and RNA, most likely due to cell shrinkage occurring in the presence of coagulant fixative. Ethanol fixation also triggers changes in composition of nucleolar proteome, as indicated by an overall reduction of the α-helical structure of proteins and increase in the concentration of proteins containing the β-sheet conformation. We conclude that cross-linking fixation is a more appropriate protocol for mapping of proteins in situ. At the same time, ethanol fixation is preferential for studies of RNA-containing macromolecules. We supplemented our quantitative Raman spectroscopic measurements with mapping of the protein and lipid macromolecular groups in live and fixed cells using coherent anti-Stokes Raman scattering nonlinear optical imaging.

    Copyright © 2014 American Chemical Society

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    Supporting Information

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    Transmission and CARS images of measured cells, raw and processed Raman spectra, background and standard error curves for cell no. 3, BCA results for time-lapse measurements, individual BCA proteins components, and PCA analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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    This article is cited by 30 publications.

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    Analytical Chemistry

    Cite this: Anal. Chem. 2014, 86, 21, 10909–10916
    Click to copy citationCitation copied!
    https://doi.org/10.1021/ac503172b
    Published September 29, 2014
    Copyright © 2014 American Chemical Society
    Request reuse permissions

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