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Correlative Microscopy on Ice

SBKB [doi:10.1038/sbkb.2014.216]
Technical Highlight - August 2014
Short description: New methods for correlated cryo-electron tomography and super-resolution fluorescence imaging are now described.

Correlated cryo-PALM-CET allows visualization of T6SS sheath in Myxococcus xanthus in red and yellow (low and high precision, respectively); cellular structures are shown in blue, green and white.

Methods to image macromolecular complexes within cells can contribute substantially to our understanding of cellular organization and function. Cryo-electron tomography (CET) is one such technique: it performs at nanometer-scale resolution, sufficient to visualize macromolecular structures and, critically, avoids fixation artifacts because it is carried out in unfixed cells. But this powerful method has a drawback: with few exceptions, the experimenter does not know which of the observed complexes in a cryo-electron tomogram contain specific proteins.

In recent work, Jensen and colleagues tackle this problem by developing methods to combine CET with super-resolution fluorescence imaging. When applied to cells expressing a protein of interest fused to a fluorescent reporter, this correlative approach allows identification of complexes containing the labeled protein. In contrast to standard correlative imaging, the super-resolved fluorescence images should permit precise identification of the complex of interest within a crowded cytoplasm.

To adapt photoactivated localization microscopy (PALM) to cryogenic conditions, the authors modified several aspects of standard protocols. Since PALM is based on high-resolution localization of fluorescent proteins that are stochastically photoactivated, the researchers first tested which probes retain their properties under cryo conditions: photoactivatable-green fluorescent protein (PA-GFP) emerged as the only usable probe.

In addition, they needed to stabilize and insulate the cryo-PALM microscope to prevent drift, which would reduce spatial resolution, and determine conditions to prevent ice crystal formation, which can occur due to warming of the frozen sample by the intense laser illumination required for PALM. They observed crystal formation only extracellularly, and were able to minimize it with a combination of cryoprotectants and a pulsed laser protocol. They speculate that the crowded cytoplasm of their bacterial sample acts as a natural cryoprotectant, but caution that this must be evaluated for every cell type.

The researchers performed correlated cryo-PALM and CET in cells of bacterium Myxococcus xanthus. They imaged a transgenic strain in which the endogenous VipA protein, a component of the bacterial type VI secretion system, was replaced by a PA-GFP tagged version. By studying only the filamentous structures that correlated with VipA fluorescence, they specifically identified the dynamic T6SS filaments in the presence of other filamentous structures, and could examine their properties in the context of the intact, unfixed cell.

Natalie de Souza

References

  1. Y. Chang et al. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography.
    Nat. Methods. 11, 737-739 (2014). doi:10.1038/nmeth.2961

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