The overall goal of this procedure is to perform a parallel plate flow chamber adhesion assay using a dual camera emission splitting system to simultaneously record realtime image sequences in two colors. To do this, the two cameras on the imaging setup are first aligned. Next, the cells and reactive substrate are prepared and the parallel plate flow chamber is set up.
The camera settings are then calibrated for two color image acquisition and any needed digital corrections are made to camera alignment. Image sequences are then captured, which demonstrate acquisition of dual color images with high temporal resolution. The advantage of dual camera emission splitting systems over single camera systems is that different exposure times can be assigned to each camera and the full field of view is retained.
This technique can help answer key questions in the field of cell adhesion, such as how might localization of adhesion molecules on the surface of cells affect dynamic biological processes such as cell migration, inflammation, and cancer metastasis. The following procedure requires an inverted epi fluorescence microscope. This should be equipped with a high intensity light source coupled to a light guide, a combination filter cube, and a dual camera emission splitting camera system.
The microscope setup should be connected to a computer running imaging software capable of collecting and combining images captured by monochrome CCD cameras. Here, stream picks five multi-camera software is used with the simul picks module. Begin by opening stream picks five in the task ribbon, select workspace.
Then at the far right of the screen, open workspace manager and select new workspace. After naming the camera, follow software prompts to select from a pre-populated list, a grabber matching camera model, a small camera icon will appear in the workspace to indicate that the camera feed is received. Objects imaged with the microscope may now be viewed on screen.
Repeat this process for the second camera workspace, then for the merged workspace, repeat this once more and all cameras are in use. Error message will appear. This message is normal assigned cameras from workspace one and two to the merged workspace in the docking panel located on the right hand side of the viewing screen.
The images in workspace one and two will be superimposed in the merged workspace as two pseudo colored images. The most difficult aspect of this procedure is camera alignment. To ensure success, we use the manufacturer slide while meticulously following the manufacturer's instructions.
To align the cameras in the dual camera emission splitting system, send brightfield or phase contrast light to the microscope eyepiece. Place the metallic coated calibration grid that was provided by the manufacturer on the microscope stage and square the grid on the microscope stage. Display the grid without binning and without auto-scaling and bring it into focus then displace, but do not remove the dichroic housing to align camera one.
Then using the focus adjustment dial on seamount port one, focus the image again. Next, push the dichroic housing into the dual channel acquisition device fully so that the image is split by the dichroic to both cameras. Loosen the 3 5 60 fourth inch set screws and rotate the second camera on female CM mount port two until the orientation of the grid is identical in both images using the focus adjustment dial on C mount port two, bring the image from camera two into focus.
To finalize the alignment, use the RL knob on the right side of DC two to adjust the combined image positions until the right lift. Vertical boundary of the images is precisely aligned in the merged workspace. Then use the UD knob to adjust the up down alignment of the second image until the horizontal grid bars are properly aligned.
If during the up down alignment a slight camera rotation occurs, correct this issue by loosening the 3 5 60 fourth inch screws for camera two and rotating until the displayed image is properly aligned. Prepare BET 20 breast cancer cells for parallel plate flow chamber adhesion assay by staining them with anti CD 24 and TECA 4 5 2 using LOR 5 68 and 4 88 secondaries as described in the accompanying document, be sure to prepare the appropriate single color controls following staining dispense cells into the reservoir of the parallel plate flow chamber. Connect two separate lengths of tubing to the inlet and outlet ports of the flow chamber.
One tube will connect to the reservoir containing the labeled cells suspended in DPBS plus containing 0.1%PSA. Connect the other tube to the syringe pump, which will withdraw fluid at a specified flow rate. Connect a vacuum line to the flow chamber and position the flow chamber over the 35 millimeter tissue culture dish containing a monolayer of Chinese hamster ovary cells expressing lectin or cho e cells.
Adjust the flow chamber and flow chamber gasket to ensure an adequate vacuum seal. Withdraw fluid from the reservoir monitoring the flow chamber assembly for proper seal. Finally, place the flow chamber assembly on the microscope power on the light source and microscope setup by manual inspection.
Ensure that the dichroic housing is in the correct depressed operating position and is not in bypass mode. Manually change to fluorescence mode and select the green red fluorescence filter cube to determine the exposure time and gain separately. Image the cells using red fluorescence in camera one and green fluorescence in camera two.
Manually depressed the dichroic as needed to obtain the images. Next deposit a suspension of BT 20 cells stained with only red flora. Four conjugated antibody in the reservoir connected to the inlet port of the parallel plate flow chamber.
Use the syringe pump to perfuse BT 20 cells over the choi monolayer in the parallel plate flow chamber. In the live settings of the imaging software, adjust the exposure time of camera one to obtain an image in the red channel. Match the exposure time of camera two to the exposure time of camera one.
If an image is observed in the green channel, bleed through artifacts are present. If this happens, keep the exposure time equivalent in camera one and camera two and reduce the exposure time until the bleed through artifact is no longer visible in the green channel. Adjust the gain of camera one to improve image visibility in the red channel if necessary.
Next, remove any remaining BT 20 cell suspension from the reservoir and replace the suspension with DPBS. Use the syringe pump to perfuse the solution over the choi monolayer until the BT 20 cells are no longer visible on the monolayer. Refill the reservoir with the suspension of BT 20 cells stained with only green fluoro four conjugated antibody.
Repeat the camera calibration process using the green single color controls. This time define exposure time with camera two to image cells in the green channel without bleed through artifacts in the red channel collected by camera one. Use the imaging software to simultaneously view images captured from the two monochrome CCD cameras in the flow chamber.Experiment.
Merge the images collected from both cameras using the simul pics module of stream picks. Use digital correction adjustments in simul pic or alternative software to spatially align the merged images. If necessary, images can be corrected with the simul PIC module for horizontal, vertical and rotational adjustments.
The system is now ready to be used to capture two color images simultaneously. A parallel plate flow chamber adhesion assay was used to demonstrate the dual camera emission splitting system described in this video. As shown here, the system detected BT 20 cells that were fluorescently labeled with anti-human CD 24 shown in red and HECA 4 52, which binds to SCOs related molecules shown in green.
Some rolling cells displayed red but not green signals. Others displayed green signals but not red signals, and yet other cells displayed both red and green signals.Here. The cameras maintained the optical and temporal resolution to track cell features on a cell tumbling and over end as it rolled on the reactive substrate in the direction of flow merged emission channels revealed the distribution and colocalization of CD 24 and sated molecules on the surface of BT 20 cells rolling and adhering to CHO e Monolayers.
These images show a zoom of a single BT 20 cell in which CD 24 and sated molecules co localize and appear as yellow to orange spots when the red and green pseudo colored emission channels are merged. Taken together. This information demonstrates that the dual camera emission splitting system has the spatial, temporal and optical resolution needed to reveal cellular and molecular features in applications such as flow chamber adhesion assays in which cells move rapidly through the field of view.
After watching this video, you should have a good understanding of how to set up and calibrate a dual camera emission splitting system or imaging a parallel plate flow chamber adhesion assay in real time in two colors. This method can be applied to other in vitro assays that use fluorescence to study cell behavior.
