Name: correlative microscopy for 3d structural analysis of dynamic interactions
Uploaded: 2013-06-24T13:00:00
Description: Watch this Scientific Journal Video about Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions at JoVE.com

The authors would like to thank Travis Wheeler and the machine shop at the Department of Cell Biology and Physiology, University of Pittsburgh for construction of the cryo-fluorescence sample stage, Changlu Tao and Cheng Xu at the University of Science and Technology of China for technical assistance, and Dr. Teresa Brosenitsch for critical reading of the manuscript. This work was supported by the National Institutes of Health (GM082251 &amp; GM085043).

1. HeLa Cell Culture on Carbon-coated, Gold EM Finder Grids
<ol>
	<li>Glow discharge the carbon-side of a 200-mesh R2/2 Quantifoil gold EM finder grid under 25 mA for 25 sec.</li>
	<li>Coat the EM grid with fibronectin, by floating it, carbon-side down, onto a 40 &mu;l droplet of 50 &mu;g/ml fibronectin solution, then disinfect it under UV light for 2 hr in a tissue culture hood.</li>
	<li>Plate HeLa cells onto the grid at a density of 2 x 104 cells/ml (total 2 ml culture) in a glass-bottom culture dish and ...

<ol>
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We have presented a straightforward set of protocols to provide an advanced correlative approach to analyze dynamic cellular events using time-lapse confocal, live-cell fluorescence imaging followed by cryoET. Our methodological development to correlate 3D live-cell imaging with high-resolution cryoET is critical to investigate many challenging biological problems, such as visualizing rare, dynamic (not static, as previously reported), and diffraction limited viral particles and their interactions with host cells. The sp...

Cryo-electron tomography (cryoET) is a powerful imaging technique that allows three-dimensional (3D) visualization of cells and tissues and provides insights into the organization of native organelles and cellular structures at molecular resolution in a close-to physiological state1. However, the inherently low contrast of unstained frozen-hydrated specimen, combined with their radiation sensitivity, makes it difficult to locate areas of interest inside a cell and subsequently performing the tilt series successfully without damaging the target area. In order to overcome these problems, a correlative approach that combines light and electron microscopy is necessary. Specific features highlighted by fluorescent labeling are identified and located by fluorescence light microscopy, and then their coordinates are transferred to the electron microscope for acquisition of high resolution 3D structural data. This correlative method helps locating the target areas of interest to be addressed. Due to the limitation on sample thickness with cryoEM (&lt;300 nm), currently only the peripheral regions of the cell are suitable for 3D structural analysis by CryoET. Further reducing the thickness of frozen-hydrated specimens by vitreous sectioning8 or by cryo-focused ion beam (FIB) milling9 would expand the capability of correlative imaging.
Previously, correlative methods were primarily used to facilitate cryoET data acquisition for large and static structures10-13. In these studies, cryo-stages have been implemented to accept cryoEM grids and fit onto either an upright microscope or an inverted microscope10,11,14. Although switching grids seems fairly straightforward in their designs, there are additional transferring steps involved for the EM grid, increasing the chance that the grid may be deformed, damaged and contaminated. We recently demonstrated a technical advance in correlative microscopy that allows us to directly visualize dynamic events that are by nature difficult to capture, such as HIV-1 and host cell interactions at early stages of infection15. We accomplished this by designing and implementing a cryo-light microscopy sample stage that adapts a cartridge system to minimize the specimen damage due to grid handling, thus facilitating correlation. Our design includes an integrated specimen cartridge holder, allowing both cryo-light and cryo-electron microscopy to be performed, sequentially, on the same specimen holder, without sample transfer, thus streamlining the correlative process. In addition, we also implemented an accurate and reliable correlation procedure using fluorescent latex beads as fiducial markers.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>DMEM 4.5 g/L Glucose w/ L-Glutamine</td>
			<td>Atlanta Biologicals, Inc. Lawrenceville, GA</td>
			<td>12-604F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma, St. Louis, MO</td>
			<td>F1141-1MG</td>
			<td>HeLa cell culture on EM grids</td>
		</tr>
		<tr>
			<td>Cell tracker</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>C34552</td>
			<td>Red CMTPX</td>
		</tr>
		<tr>
			<td>Protein A gold conjugates</td>
			<td>Ted Pella, Inc., Redding, CA</td>
			<td>15822-1</td>
			<td>15 nm diameter</td>
		</tr>
		<tr>
			<td>0.2 &mu;m fluorescent microspheres</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>F8811</td>
			<td>Yellow-green fluorescent</td>
		</tr>
		<tr>
			<td>Heat-inactivated fetal calf bovine serum</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>10082-139</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass-bottom culture dish</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gold quantifoil finder EM grids</td>
			<td>Quantifoil Micro Tools, Jena, Germany</td>
			<td>R2/2 Au NH2 200 mesh</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen Corporation, Carlsbad, CA</td>
			<td>70011-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Glow-discharge device 100X</td>
			<td>EMS, Hatfield, PA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tecnai Polara G2 electron microscope with a Field Emission Gun</td>
			<td>FEI, Hillsboro, OR</td>
			<td>&nbsp;</td>
			<td>300 keV</td>
		</tr>
		<tr>
			<td>Vitrobot Mark III</td>
			<td>FEI, Hillsboro, OR</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus IX 71 microscope</td>
			<td>Olympus America Inc., Center Valley, PA</td>
			<td>&nbsp;</td>
			<td>LUCPlanFLN 40X/0.6 NA (2.7-4 mm working distance) objective lens</td>
		</tr>
		<tr>
			<td>Nikon TiE microscope</td>
			<td>Nikon Instruments, Melville, NY</td>
			<td>&nbsp;</td>
			<td>using a 60X/1.35 NA oil immersion objective lens</td>
		</tr>
		<tr>
			<td>Sweptfield confocal microscope</td>
			<td>Prairie Technologies, Middleton, WI</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tokai Hit live cell chamber</td>
			<td>Tokyo, Japan</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryo-fluorescence sample stage</td>
			<td>Home-made</td>
			<td>&nbsp;</td>
			<td>Homebuilt by machine shop, see reference 15 for the design.</td>
		</tr>
	</tbody>
</table>

correlative microscopy for 3d structural analysis of dynamic interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, 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-13-7, 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.

To characterize the dynamic behavior of the virus particles, HeLa cells infected with HIV-1 were imaged by high-speed confocal live-cell microscopy and the particle movements were analyzed by automated 3D particle tracking (Figure 1). To avoid the time lapse of several minutes that can occur between collection of the last confocal live-cell image and plunge-freezing (which may be long enough to lose correlated HIV-1 particles), a cryo-fluorescence light microscopy stage (Figure 3) was de...

Watch this Scientific Journal Video about Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions at JoVE.com

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

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