L.W.E. and R.K. are supported by the US National Institutes of Health grants R37 NS033506-16 and R01 NS060699-03. M.P.T. was supported by an American Cancer Society Postdoctoral Research Fellowship (PF-10-057-01-MPC). The advice and knowledge of Dr. Matthew Lyman and Dr. Oren Kobiler were instrumental in designing the microscope configuration and live cell imaging methods. We also extend thanks to Neal Barlow and Brian T. Kain of Nikon Instruments for their technical assistance in the acquisition, installation and performance of the microscope.

Section 1 - Environmental Control Conditions for Live-cell Fluorescence Microscopy
<ol>
	<li>30 min prior to any imaging experiment connect and begin warming the microscope stage top incubator.
	<ol>
		<li>Turn on microscope, transmitted and epifluorescent light sources, and automated hardware controls. Open the microscope control software, NIS Elements, to connect to the microscope and begin cooling the EM-CCD camera.</li>
		<li>Attach Lens warmer to appropriate objective (either 60X or 100X oil immersion). No...

<ol>
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We have nothing to disclose.

The goal of live cell imaging is observing biological processes as they occur without significant alteration by the act of observation. This goal is achieved by optimizing three variables: environmental control, speed of imaging, and fluorescence illumination. These inter-dependent variables must be balanced to achieve viable imaging conditions. The protocols presented utilize specific conditions to produce the representative results. We will briefly discuss environmental controls and observational damage before detailin...

Infection of the peripheral nervous system (PNS) by alphaherpes viruses such as herpes simplex virus (HSV) -1, -2, and pseudorabies virus (PRV) involves several intricate and highly regulated steps throughout the viral life cycle. Transport within neurons of the PNS is critical during the events of both primary viral infection and subsequent inter-host spread. The molecular mechanisms that modulate two components of the viral life cycle; directed transport of viral assemblies away from cell bodies within axons (anterograde transport) and subsequent transmission of virions to susceptible cells (anterograde spread) are important to understanding herpesvirus pathogenesis.
The transport and egress of viral particles in neurons is dependent on assembly of a mature infectious virion 1,2. Previously fixed assays, including immunofluorescence (IF) and electron microscopy (EM), were used to study the particle assembly state and protein interactions associated with virion transport and spread 3-6. However the dynamic nature of transporting virions and experimental artifacts introduced by chemical fixation confounded the interpretation of fixed images 7,8. Recently, a number of viral-fluorescent fusion proteins have been described for HSV and PRV that have negligible impacts on protein function. Green Fluorescent Protein (GFP) and Red fluorescent proteins (mCherry or mRFP) fluorophores are often paired to allow imaging of two of the three structural components of a mature virion: capsid, tegument, and glycoprotein 9-11. Live cell imaging of anterograde transport using dual-labeled viruses visualizes the assembly state of viral particles during transport. Similar fluorophore expressing viral strains are used to visualize number and the diversity of viral genomes following spread 12,13. The properties of the fluorescent proteins (reviewed in 14,15) greatly impact the ability to visualize viral or cellular assemblies. The intrinsic properties of the fluorescent protein, including self-interactions and stability, should be considered when designing and testing novel protein fusions for the preservation of wild-type functionality.
In conjunction with fluorescent protein fusions, two well-characterized in vitro cell culture systems are employed for live cell imaging of herpes virus infection: dissociated 16 and compartmentalized 17 (Figure 1A) rat superior cervical ganglia (SCG) neurons. In both systems, embryonic SCG's are dissected, dissociated to single cell bodies, and plated for in vitro culturing 18. SCG neurons are part of the autonomic nervous system and can be readily cultured and differentiated by neuronal growth factor (NGF) into a mature polarized state ex vivo. Dissociated SCG neurons form an extended network of axons that allows for the visualization of viral particles as they undergo anterograde transport 6. Compartmentalized SCG cultures provide fluidic isolation of the neuronal cell body (S compartment) and distal axon termini (N compartment) 19. A number of detailed protocols for the construction and use of compartmentalized neurons using original 20 or modified Campenot chambers 17 have been published previously. Fluidic isolation allows for selective infection of neuronal cell bodies and detection of progeny virions after transport to isolated axon termini. Plating epithelial cells over the termini prior to infection provides recipient cells for spread of viral infection.
There are many essential elements important for all live cell imaging experimentation, but the most relevant to our protocols are: automated image acquisition, fluorescent illumination, speed of imaging, and environmental control. For all imaging experiments, we use an inverted, automated, epifluorescence illumination microscope (Figure 1B). The microscope is built around the Nikon Eclipse Ti base and employs a number of computer controlled motorized systems to rapidly reconfigure the microscope during automated image acquisition. For fluorescence illumination, we use a broad-spectrum mercury arc lamp that can be attenuated with neutral density filters along the illumination path. Excitation filters limit the spectrum of fluorescent illumination and when paired with multi-pass dichroic mirrors and fluorescence emission filters visualize specific fluorescent compounds. The excitation and emission filters are mounted in independent, fast switching filter wheels to enable rapid sequential acquisition of different fluorescent channels. The speed of image acquisition is further enhanced with a sensitive and fast EM-CCD camera, useful for detection of low intensity signals and short image read times. Environmental control on the microscope is achieved with a stage top heated incubator and an objective lens heater to maintain samples at elevated temperatures while a humidified and CO2 enriched atmosphere is passed into the incubator. The microscope is kept in a darkened room with ambient temperature maintained close to 25 &deg;C and outside light minimized with blackout curtains on all windows. The following protocols describe the use of this system for anterograde transport and spread assays.
A number of alternatives exist to control for the variables of live cell imaging. The control of microscopy settings can be performed manually or through automated systems dependent on proprietary software. Fluorescence illumination can be achieved with halogen, LED or laser sources. The speed of imaging is modified by the speed of filter switching and the time to visualize the signal with the paired detection system. Environmental control can be achieved with specialized hardware on the microscope stage, enclosure of all or part of the microscope in a heated and humidified box, or by elevating the temperature of the room where microscopy will be performed. Each of these alternatives has advantages and disadvantages related to cost and performance.
In the subsequent protocol, we detail the use of live cell imaging to study rapid anterograde transport and anterograde spread of alphaherpes viruses through the use of recombinant viral strains. Real-time live cell imaging visualizes capsid, tegument, and/or glycoprotein co-localization on dynamic structures undergoing transport within axons 11. Overnight timelapse imaging of compartmentalized neuronal cultures visualizes axonal virion egress and infection of susceptible cells 12. The protocols presented here have been optimized for use with our particular imaging system, but are presented in broad terms relative to the four elements of live cell imaging. In the discussion we will further detail some of the optimization that is necessary for successful experimentation.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>35 mm glass bottom culture dish</td>
 <td>MatTek Corporation; Ashland, MA </td>
 <td>P35G-1.5-20-C</td>
 </tr>
 <tr>
 <td>35 mm &mu;-Dish </td>
 <td>Ibidi USA LLC, Verona, WI </td>
 <td>81156</td>
 </tr>
 <tr>
 <td>Microscope body </td>
 <td>Nikon Instruments Inc. </td>
 <td>Nikon Eclipse Ti</td>
 </tr>
 <tr>
 <td>Motorized microscope X-Y stage</td>
 <td>Prior Scientific; Rockland, MA </td>
 <td>H117 ProScan Flat Top</td>
 </tr>
 <tr>
 <td>Motorized filter wheels</td>
 <td>Prior Scientific</td>
 <td>HF110 10 position filter wheel</td>
 </tr>
 <tr>
 <td>Fluorescent illumination source </td>
 <td>Lumen Dynamics; Mississauga, Ontario Canada </td>
 <td>X-Cite 120Q </td>
 </tr>
 <tr>
 <td>Multi-band Fluorescence filter sets</td>
 <td>Chroma Technology Corp.; Bellows Falls, VT</td>
 <td>89000 Sedat Quad - ET 89006 ECFP/EYFP/mCherry - ET</td>
 </tr>
 <tr>
 <td>EM-CCD camera</td>
 <td>Andor Technology USA; South Windsor, CT</td>
 <td>iXon3 897 </td>
 </tr>
 <tr>
 <td>Chamlide stage top environmental incubator</td>
 <td>Live Cell Instruments; Seoul, South Korea</td>
 <td>TC-L-10</td>
 </tr>
 <tr>
 <td>Objective lens heater</td>
 <td>Bioptecs; Butler, PA</td>
 <td>150803 Controller 150819-12-08 Heater</td>
 </tr>
 <tr>
 <td>Analysis software</td>
 <td>Nikon Instruments Inc.; Melville, NY</td>
 <td>NIS Elements </td>
 </tr>
 </tbody>
</table>

live cell imaging of alphaherpes virus anterograde transport and spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Anterograde Transport
Application of this protocol to infections of dissociated SCG cultures with PRV 348, a recombinant PRV strain expressing GFP-Us9 and gM-mCherry membrane fusion proteins, has facilitated the visualization of the anterograde transport of virions (Figure 3 and Supplemental Movie 4). Incorporation of these fusion proteins into viral particles results in their detection on transporting puncta, and the aforementioned imaging conditions minimize th...

Watch this Scientific Journal Video about Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread at JoVE.com

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

live-cell-imaging-alphaherpes-virus-anterograde-transport

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Immunology and Infection

10.9K 조회수.  Montana State University. alphaherpes 바이러스 감염의 라이브 셀 이미징 직접 수송 및 간 스프레드의 동적 이벤트 분석을 할 수 있습니다. 여기, 우리는 차 신경의 감염시 바이러스 어셈블리의 시각화를 촉진하기 위해 형광 융합 단백질을 발현하는 재조합 바이러스의 변종을 활용 방법론을 제시한다.