Imaging Protein Labels with Liquid STEM
an intact fixed cell in liquid. Currently, liquid STEM is limited to surface proteins and proteins that internalize, but strategies for labeling intracellular proteins with high-Z labels [32, 33] may be used to label intracellular proteins in the future. Fluorescence microscopy may be used to monitor tagged proteins in living cells to determine the desired time point of the fixation, such that a specific state of the cell can subsequently be studied with liquid STEM; initial results show that liquid STEM of unfixed cells is also feasible [34]. By repeating the experiment, bringing the cells into different functional states, liquid STEM can be used to study cellular function at the level of protein complexes via a direct method. Tis should lead to a broad range of applications in biomedical research.
Conclusions Liquid STEM is capable of imaging individual gold
nanoparticles, labeling specific proteins in cells, as well as QDs labels, used for correlative fluorescence and electron microscopy. Te sample preparation method for liquid STEM is similar to standard methods employed for light microscopy, with the difference that high-Z nanoparticle labels are needed to provide contrast in STEM. Te images reveal the locations of protein labels with high resolution, while the biological structure is visible with less contrast, as is the case in fluorescence microscopy. Liquid STEM has a key advantage over state-of-the-art TEM: Cells are labeled live and then fixed. Aſter this point, no further sample processing is required. Tus, artifacts oſten introduced by dehydration, post-staining, freezing, or sectioning are avoided. Liquid STEM combines some of the functionality of light microscopy with the resolution of electron microscopy.
Acknowledgments We are grateful to J. Bentley, S. Head, G.J. Kremers, and
T.E. McKnight for help with the experiments, and to Protochips Inc. for the liquid STEM system. A Portion of this research was conducted at the SHaRE User Facility, which is sponsored by the Division of Scientific User Facilities, Office of Basic Energy Sciences, U.S. Department of Energy. Research supported by Vanderbilt University Medical Center, NIH grant GM72048 (DWP), and NIH grant 1R43EB008589 (to S. Mick).
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