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). Note: Select objective lens based on experiment type. The 100X differential interference contrast (DIC) is best for tracking of anterograde transporting virion assemblies while the 60X phase objective is suited to visualize anterograde spread. Lower magnification, non-oil immersion objectives do not require a lens warmer as they do not make direct contact with the sample.</li>
		<li>Attach stage top incubator onto the motorized stage of microscope. Ensure the appropriate insert that will hold the sample is in place. Attach the humidified air line from the incubator control to the input port on the stage top incubator.</li>
		<li>Turn on incubator and lens warmer controls and adjust temperature settings to previously verified conditions specific for the objective and sample being imaged (see discussion).</li>
		<li>Open the control valve on the 5% CO2/95% atmospheric tank to provide CO2 supplemented air to stage top incubator. The flow rate should be between 60 and 80 ml of gas per minute. Faster rates will result in extensive sample dehydration despite the humidifying chamber.</li>
	</ol>
	</li>
	<li>Add lens oil to objective lens and immediately place the culture that will be imaged into the stage top incubator.</li>
	<li>Allow equilibration of temperature in the culture for a minimum of 10 min. One hour is preferred, especially for overnight imaging described in Section 3.</li>
</ol>
Section 2 - Real-time Imaging of Anterograde Virion Transport Events Protocol
<ol>
	<li>Six to eight hours prior to imaging, infect a 35 mm coverslip-bottom dish of mature, dissociated SCG neurons with 1 x 106 plaque forming units of the virus of interest (See Table 1 for example viruses) using the method described in Taylor et al. 2012a 11. If testing more than one virus, off-set inoculations by 30-60 min to allow for imaging of sequential samples at similar times post infection.</li>
	<li>Insert dish into stage top incubator for a minimum of 10 min before running any experiment. While the temperature equilibrates, the focal plane is shifting and will negatively impact any imaging experiments.</li>
	<li>Using transmitted light and the eyepiece of the microscope, find a neuronal cell body that has a clearly isolated axonal extension. Alternatively, use the appropriate fluorescence illumination to find a cell expressing detectable amounts of fluorescent proteins. It is important to limit exposure of sample to fluorescent illumination, at this step and subsequent steps, because of light induced cellular damage and bleaching of fluorescent proteins.</li>
	<li>Attenuate fluorescence illumination to reduce the intensity of excitation light to which the cells are exposed. Engage light attenuating Neutral Density (ND) filters in the fluorescence illumination light path. For movies lasting longer than 1 min, a minimum of ND4 must be used to prevent photo-bleaching of fluorescent proteins and extensive photo-damage of axons. Highly fluorescent structures can typically be visualized with an ND8 filter in place.</li>
	<li>Using the microscope software (Figure 2 and Supplemental Movie 1A), switch the light-path to the EM-CCD camera.</li>
	<li>Determine optimum imaging times and conditions for each fluorescent channel.
	<ol>
		<li>Initiate a live image window using the "Play" button.</li>
		<li>Determine the optimal camera exposure settings for each fluorescent protein. Do not exceed 300 msec per channel as motional aberrations of fast moving virion structures will occur. Utilize the EM gain of the camera to a setting of 300 (manufacturer suggested maximum gain), to minimize exposure time and enhance detection of dim signals. Correct exposure times should produce an image with low background intensity and high specific signals.</li>
	</ol>
	</li>
	<li>After setting exposures and finding a well-defined region of axon, the software must be configured to perform automated image acquisition. Stop the live imaging window to prevent sample bleaching.
	<ol>
		<li>In the NIS Elements software, select the 6D - Define/Run Experiment application from the "Applications" drop down menu.</li>
		<li>In the ND acquisition window, click the Time tab which will determine the frequency of image acquisition. Set the Interval to "No Delay" and the Duration to 5 min. The software will then acquire image frames at the highest frequency possible for duration of 5 min.</li>
		<li>Select the "Lambda" tab, which allows selection of the pre-set hardware configurations used for multiple fluorescent image acquisition. Click the first box and in the subsequent drop down menu to select an appropriate hardware configuration. Repeat for all fluorescent proteins to be imaged.</li>
		<li>Ensure the tabs for "XY Positions", "Z-series" and "Large Image" are de-selected.</li>
		<li>Above the tabs, select the box marked "Save to File" to automatically save images to the hard disk during acquisition. Configure the location and file name of the experiment output file. As sequential experiments are performed, the software will add a sequential number at the end of the designated file name.</li>
		<li>Once all tabs have been selected. Initiate the experiment by clicking the "Run-Now" button.</li>
		<li>Optimize the image acquisition rate through exposure settings and image size. Using a small region of the image-able area will often speed up frame acquisition rates. Define the Region of Interest using the ROI tool in NIS-Elements, selecting an area between 25% and 50% of the total image area. Alternatively, shorter exposure times will increase frame acquisition rates, but decreases the quality of signal (see discussion).</li>
	</ol>
	</li>
</ol>
Section 3 - Overnight Time-lapse Imaging of Anterograde Spread Events
<ol>
	<li>Four hours prior to imaging, infect a compartmentalized neuronal culture constructed on a 35 mm &mu;-Dish. Inoculate neuronal cell bodies with 1 x 106 plaque forming units of the virus of interest (See Table 1 for example viruses) using the method described in Taylor et al. 2012b 12.</li>
	<li>Gently place culture dish into the stage top incubator. Ensure the position of the objective is set to the side of the dish. Slowly bring the objective into focus, ensuring the objective does not push up into the sample. Once the focus field has been identified, slowly move the objective into the center of the dish, near the internal barrier demarking the internal side of the axon compartment. During movement, ensure the focus depth is maintained and avoid pushing up into the sample. Deflection of the plastic surface by the objective will damage the axons and the plastic.</li>
	<li>Add approximately 2 ml of warm (~37 &deg; C) phosphate buffered saline to the culture dish outside of the Teflon ring. This surrounds the neuronal culture with a ring of PBS to minimize sample dehydration and provide thermal insulation.</li>
	<li>Allow temperature to equilibrate for a minimum of 1 hr prior to initiating automated image acquisition (3.8). Steps 3.5 through 3.7 can be configured during this time.</li>
	<li>Restrict the intensity of illumination to the lowest possible level by engaging all neutral density filters. This will allow frequent image acquisition over long time periods without excessive photo-damage. Reducing illumination will result in low signal, requiring longer exposures (see discussion).</li>
	<li>Use historical exposure settings or prior to experimental set-up generate a parallel dish of infected epithelial cells expressing the target fluorescent proteins to establish exposure settings as described in Section 2.5.</li>
	<li>After setting exposures for the necessary channels, it is time to configure the software for automated image acquisition. Stop the live imaging window to limit sample bleaching.
	<ol>
		<li>In Elements software (Figure 2 and supplemental movie 2), select the 6D - Define/Run Experiment application from the "Applications" drop down menu to bring up the ND Acquisition window.</li>
		<li>In ND Acquisition window, click the Time tab. Set the Interval to "5 min". Set the Duration to 20 hr. The loops field will be automatically calculated for the number of repetitions the program will perform.</li>
		<li>Select the "Lambda" tab. Click the first box and in the subsequent drop down menu select the appropriate lambda configuration for Phase contrast. Repeat for subsequent positions, selecting the appropriate configurations for each fluorescent protein to be imaged.</li>
		<li>Select the "Large Image" tab, which determines the parameters of multiple images to be combined into a single larger field of view. A good-sized area consists of 5 x 2 images using a 7% stitching overlap. This will allow for the maximum number of cells per position to be imaged without impacting the frequency of image acquisition. Also, deselect the checkbox for "Close Active Shutter during Stage movement". This option enhances the speed of acquisition by not closing shutters between images.</li>
		<li>Select the "XY Positions" tab, to identify multiple positions that will be sequentially imaged in parallel during the course of the overnight movie. Select positions that define the center point for each large image area. Select 6-8 image positions in the sample. A good image area will have small clusters of cells in close apposition to clearly defined axons. Cells should not be piled on top of each other and axon bundling should be minimized. The point of focus is the nucleus of the cell. Position the objective such that a clearly defined nuclear envelope is seen for the majority of the cells.</li>
		<li>Ensure the "Z-series" tab is deselected.</li>
		<li>Configure the auto-save feature as described above in Section 2.6.5.</li>
		<li>Test the exposure and position settings by clicking "1 time loop". Evaluate the resulting images for transmitted light illumination, focus, and framing. If focus has shifted, reset the focus for each position under the X-Y tab. Wait 10 min and repeat this step until focus is stable.</li>
		<li>After all settings have been verified and tested, initiate the automated experiment by clicking the "Run Now" button.</li>
	</ol>
	</li>
</ol>
Section 4 - Image Processing and Export
Data Export
<ol>
	<li>Datasets obtained during live-cell imaging of transport or spread events should be exported from the NIS Elements software into commonly used file types for later use in presentations and publications (Supplemental Movie 3). To export movies open the appropriate raw .nd2 file in NIS Elements and select the "split-component" view to visualize the desired fluorescent channels. Playback the file at a suitable speed; 5 frames per second is a good starting point for visualization of fast-transport.</li>
	<li>Include a timestamp to represent a real-time clock during movie playback. Right-click on the image and select, Add ND-information. A digital counter will appear in the upper left corner.
	<ol>
		<li>Edit the time-stamp to display information. Right click on the counter and select Edit ND-information. In the subsequent pop-up window, edit the text to reflect the imaging parameters that are important. Edit font, color and size for clarity.</li>
	</ol>
	</li>
	<li>Go to the "Edit" menu and find "Create view snapshot" or press the "x" key. This command opens the "view snapshot" window. Select "apply to all frames" to generate an 8-bit, RGB, export-ready file containing the fluorescent channels from the entire movie.</li>
	<li>Save as an .avi file for cross-platform compatibility. Set playback rate at 200 msec per frame. The .avi files generated by NIS Elements can then be further compressed with other conversion software into the desired file type.</li>
</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

Research

JoVE Journal

Immunology and Infection

11.0K Views.  Montana State University. 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.

Video: Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...