The overall goal of the following experiment is to use optical trapping combined with spinning disc confocal microscopy to observe host pathogen interactions in real time. This is achieved by first adding the pathogens of interest to a chambered cover glass. Next, a particle is selected and captured with the optical trap.
Finally, the particle is directed to the cell of interest. As seen here. Optical trapping can be an effective method for controlling two particles for observation of dynamic intracellular interactions.
This method can answer important questions in host pathogen interactions in the fields of infectious disease and immunology, including what is the effect of on phagocytosis when complete control of spatial relationships between host cells and pathogens are obtained, as well as whether the order of pathogens being taken up by a phagocytic cell affect pha somal maturation. Generally, individuals new to this method will struggle because trapping conditions have to be optimized for different cell and bead types. Visual demonstration of this method is critical as the process to set up and integrate a spinning disc.
Confocal to an optical trap is difficult to conceptualize due to the precise alignment that is necessary for all the optical components. Harvest the pathogen of interest. For example, here, 300 microliters of Canada albicans grown overnight in broth is removed from culture and transfer the pathogens into a 1.5 milliliter reaction tube.
Then wash the pathogens three times at approximately 1300 Gs for five minutes at room temperature, aspirating the supernatant and leaving the pellet undisturbed each time resuspended PBS and sonicate. After the third wash, resus suspend the pellet in 500 microliters of PBS. Next, dissolve one milligram of the dye of entrust in 100 microliters dimethylformamide for a final concentration of 10 milligrams per milliliter.
Then add three microliters of the dye mixture to the reaction tube containing the washed pathogens. Place foil around the tubes since the dyes light sensitive and shake the sample at 37 degrees Celsius for one hour, and then pellet and wash the sample in PB S3 times as before. Resuspend the pellet in 300 microliters of PBS after the last wash.
Warm media tryin and PVS to 37 degrees Celsius in a water bath Wash. A 10 centimeter tissue culture plate coated with raw 2 6 4 0.7 macrophages two times with PBS aspirating the PBS between each wash. Next, cover the plate surface with five milliliters of trypsin and incubate the plate for five minutes at 37 degrees Celsius.
Then gently knock the side of the plate to detach the cells from the plate surface. Being careful not to splash the trypsin outside of the plate. Now add five milliliters of media to the plate and transfer the mixture to a reaction tube.
After pelleting, the cells aspirate the media being careful not to disturb the pellet and resuspend the pellet in 10 milliliters of media. Add 400 microliters of media to each chamber of a chamber slide, and then add five microliters of the cell suspension to each chamber. Allow the cells to grow overnight in an incubator at 37 degrees Celsius with 5%CO2.
Retrieve the chamber slide from the incubator pipette five to 10 microliters of the previously prepared fluorescently labeled pathogens of interest into each chamber of macrophages. Mix the macrophages and pathogens thoroughly by pipetting up and down, being careful not to touch the bottom of the chamber and disturb the adhered macrophages. Turn on all the components of the spinning disc confocal microscope.
Prepare the microscope by adding oil to the oil immersion objective lens. Insert the chamber slide into the specialized stage and then remove the top of the chamber slide align microscope for DIC imaging. Turn on the shutter for the optical trap and the IR laser.
Then open the shutter on the laser to the optical trap. Confirm that the shutter in front of the IR laser is closed by checking with the IR card. Now focus on the macrophages on the adhered slide.
Then find the pathogens freely floating in the solution adjacent to macrophages. The most difficult part of this procedure is finding the right object to track in the sample chamber due to the adhesion forces between the object and the cover glass in the sample chamber. In order to move that object, you have to move the stage while the object is held by the traffic laser, and it's also important to note that you have to move the stage at slow enough loc such that the drag force of the stage does not exceed the maximum trapping force by the trap.
Move the stage such that the pathogen is in the vicinity of the trap. Then open the shutter and engage the trap. Move the sample to bring the macrophages into contact with a stationary trapped pathogen image.
The pathogens with a spinning disc confocal microscope either in DIC fluorescence or a combination of both separate populations of Canada albicans, which are typically about five microns in size, were labeled green, blue, and red. To illustrate imaging, a single C albicans was trapped and moved in a square pattern through a cluster of other yeast as indicated by the gray arrows, demonstrating the ability to capture and manipulate the specific location of a single pathogen chosen by the operator even in a crowded environment. To illustrate further the flexibility of this system to trap the different shape morphologies exhibited by pathogenic organisms in this figure, the optical tweezer was used to hold and situate a C albicans particle with a pseudo hyphy.
The C albicans is labeled with red dye and moved along a trajectory as outlined by the white arrow and placed next to fluorescent GFPL C3 raw cells. In green. The yeast portion of the albicans was trapped as the pseudo hyphy trailed along.
Aspergillus fum goddess also was positioned next to a raw mouse macrophage cell in order to analyze the absolute timeframe of phagocytosis with this particular cell line and pathogen. In this figure, bright field images show how the trapped aspergillus, as indicated by the white arrow, was moved and positioned along the path as indicated by the red arrow. The trapped pathogen is slightly outta focus due to the trap pushing the organism slightly above the focal plane.
Aspergillus was moved until it was placed adjacent to the desired raw cell. Once the pathogen made contact with the cell, the trap was turned off. The phagocytosis process was activated and time-lapse imaging was employed to observe the subsequent cellular events.
At 30 seconds, the membrane of the raw cell started to change and to form a cup around the particle. At 60 seconds, the cup was fully formed. From 90 seconds to 150 seconds, the Afu Gotti was becoming and engulfed, and by 180 seconds the particle was fully internalized.
After watching this video, you should have a good understanding on how to create a spinning disc optical trap apparatus, and how such an instrument can enable one to non-invasively control pathogens for live cell imaging After its development. This technique paved way for researchers in the field of host pathogen interactions to investigate the role of spatial relationships between host cells and pathogens, and its role on the ensuing immune response.
