The overall goal of this procedure is to demonstrate the usage of the correlative fluorescent light and cryo-electron microscopy method by imaging dynamic small HIV one particles interacting with host hela cells. This is accomplished by first plating and growing heela cells on the gold finder EM grid in a glass bottom culture dish and infecting heela cells with GFP labeled HIV one particles. The second step is to collect time-lapse high speed 3D live cell confocal images of HIV one infected cells, and analyze the images by automated 3D particle tracking for single particle dynamics after vitrify.
The sample, the same viral particles from the live cell imaging data are located in cryo fluorescent images for further high resolution 3D cryo-electron tomography analysis. The final step is to collect tilted cryo EM projection series for all of the identified positions containing HIV one particles and reconstruct 3D toms with a weighted back projection algorithm using imad software. Ultimately advanced 3D correlative live cell fluorescence microscopy and high resolution cryo-electron tomography are used to directly visualize dynamic diffraction limited viral particles and their interactions with host cells.
Our work is aimed at developing a correlation method that permits direct visualization of only events of HIV one infection By combining live cell fluorescent like microscopy, cryo fluorescent microscopy and cryo-electron tomography in this manner, live cell and cryo fluorescent signals can be used to greatly guide the sampling in cryo-electron tomography. Furthermore, structural information obtained from cryo-electron tomography can be complemented with the dynamic functional data available through lifestyle imaging of fluorescent labeled targets. The Main advantage of this technique or existing method is that advanced correlative high speed 3D lifestyle imaging with high resolution 3D coral electron tomography approach is applied to study dynamic events that are by nature difficult to catch, such as HIV type one and host cell interactions at the early stage of infection.
To prepare the grid for cell culture glow discharge the carbon side of a 200 mesh R two over two quanti foil, gold EM finder grid under 25 milliamps for 25 seconds. Coat the EM grid with fiber nin by floating it carbon side down onto a 40 microliter droplet of 50 micrograms per milliliter fiber nin solution. Leave it in a tissue culture hood under UV light for two hours for disinfection plate hela cells onto the grid at a density of two times 10 to the fourth cells per milliliter in a glass bottom culture dish.
In DMEM with appropriate supplements, incubate cells at 37 degrees Celsius with 5%carbon dioxide for approximately 18 hours before HIV one infection prior to HIV infection, add a fluorescent cell tracker to the hela cells in the glass bottom culture dish and incubate the dish at 37 degrees Celsius for 10 minutes to allow uptake of the fluorescent dye, wash the cells with PBS and add 50 microliters of prewarm fresh medium. Place the dish into the live cell chamber at 37 degrees Celsius of a swept field confocal scanner microscope. Since cryo EM requires relatively thin regions of the sample.
Select multiple fields that contain one to three cells per square with the cells between two mitosis phases when they're most spread out and flat store these positions with NIS element software for future imaging. Next, infect the cells with VSVG, pseudo typed HIV one particles that contain G-F-P-V-P-R and the virus particles into the bottom well of the dish, being careful not to disturb the EM grid. Since the positions for imaging have already been selected and stored immediately after addition of the varians collect time-lapse high speed 3D confocal images at the previously selected positions for 20 to 40 minutes.
Immediately after confocal live cell imaging, place the culture dish onto an ice cooled copper block and transfer it to the cryo EM sample preparation room. Load the EM grid onto the specialized tweezers, quickly blot away residual culture medium with filter paper immediately place onto the grid for microliters of a 15 nanometer gold bead solution mixed with 0.2 micron fluorescent microspheres. The fluorescent microspheres are used to aid correlation between fluorescent and cryo-EM.
Load the tweezers onto the vitrification device for plunge freezing with the blotting parameters optimized to achieve the best results. To begin the procedure for cryo fluorescence light microscopy connect a dry nitrogen gas line to a sleeve placed over the objective lens to keep the lens warm and free of frost mount a home-built cryo fluorescence sample stage onto an inverted fluorescence light microscope. Connect the liquid nitrogen inlet of the cryos sample stage to the self pressurized doer and place the liquid nitrogen overflow protection outlet into an appropriate container.
Place the frozen hydrated sample grid into the EM specimen cartridge on the copper block, and place a pre-cool C clip ring on top to keep the grid in place. Place the cartridge in the inner chamber of the cryo stage search and find the same virus particles from the live cell imaging data. Since the EM grid has an index, it is straightforward to localize the same particle on the grid in both live cell images and cryo fluorescence images.
Acquire cryo DIC and fluorescence images at the identified positions under cryo conditions using a light microscope with a long working objective lens. During the cryo fluorescence imaging, periodically check the liquid nitrogen level in the cryo stage and refill it with a self pressurized doer as needed to keep the sample stage below negative 170 degrees Celsius for cryo-electron tomography. Load the sample grid into the cryo transfer station of an electron microscope equipped with a field emission gun under low dose search mode at a magnification of 140 x.
Identify the regions or grid squares where cryo fluorescence images have been acquired. Record a low magnification cryo EM image of the correlated area and save the position into a stage file under a low dose search mode at a magnification of 3, 500 x. Insert a 100 micron objective aperture search and save all the positions correlated with GFP signals into a second stage file.
Set up the tomography parameters for acquiring a tilt series, including tilting angle range, electron dose, defocus value, and tilting schemes. Acquire tilt series for all the saved positions to begin this procedure. Configure the main window such that several stages of the Tomo Graham computation can be adjusted or followed simultaneously, modify the necessary parameters and execute the specific programs that are required by each processing step.
At the final stage, create a full aligned stack with a main residual error less than 0.6 and reconstruct Toms using a weighted back projection algorithm. In Im mod to characterize the dynamic behavior of virus particles. Hela cells infected with HIV one were imaged by high speed confocal live cell microscopy and the particle movements were analyzed by automated 3D particle tracking.
In this representative figure, a single green fluorescent viral particle indicated by the yellow arrowhead was tracked in 3D confocal stacks with a three minute time interval between frames to avoid the time lapse of several minutes. That can occur between collection of the last confocal live cell image and plunge freezing. A cryo fluorescence light microscopy stage was designed and constructed for imaging frozen hydrated samples within cryo EM cartridges.
On light microscopes panel A shows a self pressurized doer filled with liquid nitrogen that is used to cool down the copper cryos sample stage. Panel B shows the top inside view of the cryos sample stage with the inner chamber indicated by the white arrowhead. The sample grid is placed onto the E EM specimen cartridge, which sits in the center of a copper block platform indicated by the black arrow.
This platform was added for use with non-polar electron microscopes. Once loaded with the sample grid, the cartridges transferred to the inner chamber for cry fluorescence light. Microscopy panels C and D show respectively the specimen cartridge before and after placing a copper ring to keep the grid in place for use with a non-polar cryo-electron microscope.
The utility of the procedure for advanced correlative life cell microscopy and cryo-electron tomography is demonstrated with direct visualization of fluorescently labeled HIV V one particles interacting with a host heela cell. A DIC image recorded with a cryo light microscopy stage shown in panel A is overlaid with a cryo fluorescence image of GFP tagged particles shown in panel B.The tagged particles are colored. Red panels C, D, and E are low dose cryo-EM images of the region containing a fluorescent particle circled in panels B and C at 140 x 3, 500 x and 27, 500 x respectively.
The inset in panel E is an enlarged view recorded after acquisition of the tomographic tilt series panels, FG and H show three four nanometer thick tomographic slices separated by a distance of 21 nanometers in the Z direction. Connections between the particle and helo cell membrane are indicated by arrows in both the projection image in the inset and panel E and tomographic slices. In panels F and g.
We have presented a straightforward set of protocols to provide an advanced correlative approach to analyze dynamic virus and sterile interactions using time lapse confocal lipo cell fluorescence imaging followed by cryo-electron tomography. While attempting this procedure, it's important to remember that precise and reliable correlation between fluorescent image and the electron microscopy image is critical to successful acquisition of crow electron tomography. Data this procedure described will be useful for numerous applications involving dynamic cellular processes in cell biology such as cell signaling surface receptor internalization.
