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J Vis Exp. 2013 Jun 24;(76). doi: 10.3791/50386.

Correlative microscopy for 3D structural analysis of dynamic interactions.

Author information

1
Department of Structural Biology, University of Pittsburgh School of Medicine, USA.

Abstract

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state(1). However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging(2), due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-1(3-7), the resolution afforded by live-cell microscopy is limited (~200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target. In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

PMID:
23852318
PMCID:
PMC3728906
DOI:
10.3791/50386
[Indexed for MEDLINE]
Free PMC Article

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