The overall goal of this procedure is to visualize events of herpes virus transport and spread using fluorescent protein fusions in primary neuronal cultures. This is accomplished by first establishing sample incubation at physiologically relevant temperatures on a microscope capable of fast switching fluorescent illumination. In the second step, the parameters of the fluorescence imaging are set up to limit any cellular photo damage while acquiring reasonable data.
Next, either sequential acquisition of the fluorescent channels is performed or multi-position. Multi-image tiled frames are acquired in the final step. The data is analyzed and reformatted for presentation and publication.
Ultimately, this method of live cell imaging is used to track the movement of fluorescent viral particles within neurons and to follow infection as it spreads between cells. This method can provide insight into the transport and spread of herpes virus neurons. It can also be applied to other dynamic events in cell biology and microbiology, such as the endocytic uptake of particles or cell migration.
Before any imaging experiment, connect and begin warming the microscope stage top incubator. After 30 minutes, turn on the microscope, the transmitted and epi fluorescent light sources, and the automated hardware controls. Then open NIS elements, the microscope control software to connect to the microscope.
Here a screenshot of a standard user interface in NIS elements is displayed. The play and stop buttons control the live image window depicting the current hardware configurations for imaging, as well as what is currently being imaged. Across the top panel are a series of optical configurations that implement the preset hardware settings to configure the microscope for trans illuminated or fluorescence detection.
The camera control and exposure settings allow configuration of camera detection to optimize the image quality and speed. All the experiments are coordinated using the ND acquisition controls. Next, attach a lens warmer to the appropriate oil immersion objective, and then attach the stage top incubator to the motorized stage of the microscope.
Ensure that the appropriate insert that will hold the sample is in place, and then attach the humidified airline from the incubator control to the input port on the stage top incubator. Turn on the incubator and lends warmer controls, and then adjust the temperature settings to the previously verified conditions specific for the objective and sample being imaged. Finally, open the control valve on the 5%carbon dioxide 95%atmospheric tank to provide carbon dioxide supplemented air to the stage top incubator.
To image enter a grade virion transport events in real time. First, insert a cell culture dish containing infected neurons into the stage top incubator for a minimum of 10 minutes before running the experiment. Then using transmitted light and the eyepiece of the microscope, find a neuronal cell body that has a clearly isolated axonal extension.
Finding imageable areas in the neural culture is the most difficult part of this procedure. Success is insured by seating multiple cultures in the event of poor cell growth or infection, or physically disrupting the culture during imaging. Now use the microscope software to switch the light path to the EM CCD camera, and then click on the play button to initiate a live image window.
After determining the optimal camera exposure settings for each fluorescent protein set, the electron multiplication gain of the camera to 300. To minimize the exposure time and to enhance the detection of dim signals. Stop the live imaging window to prevent sample bleaching.
Next, after setting the exposure times and finding a well-defined region of the axon in NIS elements, configure the software for sequential image acquisition. Open the applications dropdown menu and select the six D define run experiment application. In the ND acquisition window, click the time tab to determine the frequency of image acquisition.
Set the interval to no delay and the duration to five minutes. Now click on the Lambda tab to select the preset hardware configurations used for multiple fluorescent image acquisition. Click the first box and then in the subsequent dropdown menus, select an appropriate hardware configuration.
Select the save to file box and configure the location and file name of the experiment output file. Once all these parameters have been set, click on the run now button to initiate the experiment. The image acquisition rate is optimized through the exposure settings and size of the image acquired.
Use the region of interest tool in NIS elements to define the region of interest. Selecting an area between 25 and 50%of the total image area to perform overnight time lapse imaging of enter a grade spread events. First, gently place the culture dish into the stage top incubator.
After ensuring the position of the objective is set to the side of the dish, slowly bring the objective into focus, taking care that it 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, de marking the internal side of the axon compartment, taking care to maintain the depth focus and to avoid pushing up into the sample. Next, surround the neurons in the culture dish with approximately two milliliters of 37 degrees Celsius PBS outside of the Teflon ring to minimize any sample dehydration and to provide thermal insulation.
Then engage all the neutral density filters to restrict the intensity of the illumination to the lowest possible level, allowing frequent image acquisition over long time periods without excessive photo damage. In the ND acquisition window, click the large image tab and then select an area consisting of five by two images. Using a 7%stitching overlap to sequentially image multiple positions in parallel.
During the course of the overnight movie, select the XY positions tab. Then define the positions that will define the center point for each large image area. The point of focus should be the nucleus of the cell position the objective such that a clearly defined nuclear envelope is seen for the majority of the cells.
Then click one time loop to test the exposure and position settings and evaluate the resulting images for transmitted light, illumination, focus, and framing. After all the settings have been verified and tested, initiate the automated experiment by clicking the run now button to export movies obtained during the live cell imaging. First, open the appropriate raw ND two file in NIS elements and select the split component view.
To visualize the desired fluorescent channels playback the file at a suitable speed. Five frames per second is a good starting point for visualization of fast transport to include a timestamp to represent a real-time clock during the movie playback. Right click on the image and select ADD ND information.
A digital counter will appear in the upper left corner to edit the timestamp right click on the counter and select edit ND information. In the subsequent pop-up window, edit the text to reflect the relevant imaging parameters. Edit the font color and size of the text for clarity.
Then go to the edit menu and select Create view snapshot to open the view snapshot window to generate an eight bit RGB export ready file containing the fluorescent channels from the entire movie select apply to all frames. Finally, save the file in the A VI format for cross-platform compatibility and set the playback rate at 200 milliseconds per frame. In this phase contrast image, a representative, mature dissociated, superior cervical ganglia or SCG culture employed for intergrade virion transport imaging is shown pseudo rabies virus or PRV 3 48 infected neurons express GFPS nine and GMM cherry membrane fusion proteins.
In this figure representative live cell imaging of SCG axons at eight hours post infection with P RV 3 48 is demonstrated anterograde transporting structures within distal axons are visualized in two fluorescent channels, GFP and mCherry. An anterograde transporting structure as indicated by the white arrows progresses within the axon. The two fluorescent proteins are spatially offset due to the sequential acquisition of the fluorescent channels and the rapid movement of the viral structures.
Application of this protocol to infections of dissociated SCG cultures with PRV 3 48 has facilitated the visualization of the anterograde transport of Vons. Incorporation of these fusion proteins into viral particles results in their detection on transporting puncta as visualized by the moving fluorescent dots. The aforementioned imaging conditions minimize the offset of each fluor on a moving particle during filter switching.
Utilizing fast switching filter wheels and paired multipass dichroic mirrors allows rapid sequential acquisition of two or more fluorescent channels at speeds of up to 0.8 frames per second in a three minute imaging window. Numerous transporting Punta are commonly observed and large sample sizes for colocalization analyses are rapidly generated. Here a schematic of the compartmentalized culture system pictured in this image with SCG neurons plated in the left compartment and with axonal projections extending beneath the internal barriers into the far right compartment is shown.
The following images were selected from a large image tiled time-lapse movie of virion transmission from PRV 4 27 infected axons into recipient epithelial cells. In this first panel emerged image of the transmitted YFP and RFP fluorescent images at a time post-infection. When the recipient cells are beginning to express a membrane bound YFP and VP 26 MRFP fusion proteins are shown.
The white box highlights an area of the recipient epithelial cells containing infecting capsid assemblies. Here, the same area as in the previous image as viewed through the RFP channel only is shown. The solid white line denotes schematic representation of the cell outline.
The hashed white lines indicate the cell nuclei and the blue lines outline the axons. In this second panel, the YFP and RFP channels only are shown together. Note the large number of MRFP puncta associated with the axon tracts.
These cells were recently infected and do not express either fusion protein. Infected cells can be tracked backwards to visualize the fluorescent capsids that initiated the viral infection. These individual cells can be isolated from the large frame and tracked without a significant loss of detail.
While attempting this procedure, it's important to remember that live cell microscopy impacts a biological process being observed. However, careful optimization of these protocols allows visualization without alteration.
