This method can help answer key questions in the study of immune responses to airborne pathogens about the dynamics of immune cells'interactions during respiratory infections. The main advantage of this technique is the ability to obtain bright and stable 4D images of the tracheal epithelium throughout the simple surgery. Though this method can provide insight into the neutrophil and the lytic cell interactions during influenza infection, it can also be applied to the study of different immune cells or airborne pathogens.

Generally, individuals new to this method will struggle with maintaining a stable and minimal inflamed surgery. Moreover, image analysis can be tricky due to the presence of heart defects. For intranasal influenza infection, place an anesthetized six-to eight-week-old CD11c-YFP mouse in the supine position, and confirm deep sedation by lack of response to toe pinch.

Placing the pipette close to the left nostril, dispense small droplets into the nasal cavity approximately 20 seconds apart, until the mouse has inhaled the entire 15 microliter volume of PR8 inoculate. After two to five minutes, deliver 15 microliters of virus into the right nostril in the same manner. When all of the inoculum has been delivered, confirm that the mouse is breathing normally, and place the animal in its cage in lateral decubitus with monitoring until full recovery.

Three days post-infection, remove both femora and tibiae from a transgenic CFP-expressing mouse, and gently clean the bones with forceps. Cut the bone epiphysis and use a one-milliliter syringe equipped with a 26 gauge needle to flush the bone marrow from each bone with sterile cold PBS into a 50-milliliter tube on ice. When all of the bone marrow has been harvested, aspirate the marrow through the needle tip several times and filter the dissociated cell suspension through a 40-micrometer strainer.

Collect the cells by centrifugation, and resuspend the pellet in two milliliters of cold PBS. Next, carefully layer two milliliters of each of three Percoll gradients in a 15-milliliter tube from most to least concentrated. Then carefully add two milliliters of bone marrow cell suspension onto the top gradient layer for centrifugal density gradient separation.

At the end of the centrifugation, carefully remove the band from the 64 and 72%gradient interface and wash the neutrophils in cold PBS. Then resuspend the pellet in 100 microliters of fresh PBS on ice for counting, and resuspend the cells at a five times 10 to the sixth cells per 100 microliters of PBS concentration for intravenous injection into the inoculated CD11c-YFP recipient animals. 12 hours after adoptive transfer, place the anesthetized recipient animal on a customized surgical board over a 37-degree Celsius heated surface, and remove the hair from the neck of the mouse by shaving and a depilatory cream.

Place the mouse above the plastic mouse positional with the head outside of the positional at an angle that facilitates intubation of the trachea, and secure the forelimbs, paws, and the tail with surgical tape. Apply ointment to the eyes and disinfect the exposed skin. Next, make a one-centimeter incision along the medial axis of the neck between the upper chest and the line passing through the lower point of the mandible, and laterally move the skin patches and the salivary glands to visualize the trachea.

Use forceps to carefully dissect the trachea, intubate the mouse with a catheter, and immediately start the artificial ventilation. Use a surgical hook connected to a rod on the surgical board to fix the catheter height and orientation, and expose the trachea up to the chin. Encircle the trachea with petroleum jelly and hydrate the trachea with a few drops of warm PBS.

Then glue a coverslip onto a metal holder, and screw the holder to the XYZ translator to allow the coverslip to be placed on top of the surgical preparation. It is very critical to properly expose and extend the trachea, and to lightly press it with the coverslip, to maintain the tissue stable long enough for the imaging. For time-lapse imaging in vivo, place the surgical board inside the 37-degree Celsius incubation chamber of the two-photon microscope, and add a drop of water to the coverslip.

After centering the setup and focusing on the tracheal tissue, set the scanning frequency to 800 hertz with 520 by 520 pixels, a field of view of 440 by 440 micrometers, and a line average of one. Set the titanium sapphire lasers to the appropriate wavelength and power percentage according to the excitation fluorophores, and set up the 3D and time-lapse acquisition mode with simultaneous excitation. Then define a range of 50 micrometers along the z-axis with a step size of three micrometers, and record images every 30 seconds for 30 minutes.

Following this experimental setup, stable 4D images can be captured in situ within the infected trachea over a 30-minute tracking period. Analysis of the 4D images reveals significant differences between dendritic cell and recruited neutrophil movement with a significantly faster speed demonstrated by the latter. The complex morphology of the dendritic cells causes frequent errors in cell tracking, which in turn produces tracks with a decreased duration and an increased variance in the measured directional behavior.

Therefore, a robust metric was created for measuring the directionality by considering the track duration, revealing a significant difference in the directionality of neutrophils compared to dendritic cells. In addition, computation of the distance between the neutrophils and the dendritic cells allows the detection and analysis of their contacts over time, with some neutrophils in this representative experiment forming multiple brief contacts with dendritic cells and some not forming any contact with dendritic cells during the imaging period. Moreover, study of the average trend of the distance between neutrophils and dendritic cells over time facilitates the study of the overall positioning of the cells, while investigation of the trend in specific cells allows characterization of the behavior of each individual cell.

Knowing this procedure, other methods like flow cytometry of the trachea can be performed to assess the quantification and characterization of different immune cells'recruitments in infected trachea. Don't forget that working with any influenza virus strain, even if mouse-adapted, can be hazardous, and that therefore, every procedure should be performed under biosafety level two conditions.