The overall goal of this procedure is to perform long-term microscopic imaging and tracking of neutrophils in vascular structures within the murine bone marrow through an implanted chronic window chamber over the femoral bone. This method can help answer key questions in immunology and oncology about the role of monocytes in the regulation of an infection, tumorigenesis, and tumor metastasis. The main advantages of this technique are that it enables long-term microscopic access to the femoral bone marrow, a central site of hematopoiesis, and provides dynamic information otherwise difficult to obtain by conventional methods.

Though this method is specific to neutrophil imaging within a vascular network, it may also be used to visualize other immune cell populations of interest. On the day of the surgery, confirm the appropriate level of sedation by lack of response to a toe pinch of an athymic nude mouse. Then, place the mouse on an electric heating pad, covered with a surgical drape, under a stereo microscope.

Next, apply ointment to the animal's eyes, and sterilize the surgical area with two sequential wipes with 7.5%povidone-iodine, 70%isopropyl alcohol, and 10%povidone-iodine. When the animal has been prepped, use a scalpel with a number 15 blade to make a 10 millimeter longitudinal skin incision approaching the femur from the lateral side. Then, blunt dissect with forceps and hemostats to expose the femur between the flexor and the extensor muscles.

When the femur can be observed, insert a U-shaped bar under the bone, and install the window chamber. Using two nuts, secure the bar to the window. Then, tighten the nuts with forceps.

Use the forceps to readjust the musculature of the flexors and extensors back to their original locations. Fill the space between the bone and the window chamber with dental cement to fix the window chamber in place. Then, use a micro drill to grind a six by two millimeter area into the cortical bone to gain optical access to the marrow cavity.

When an almost transparent periosteal layer of the bone has been obtained, place an eight millimeter cover glass onto the window, and use a snap ring to secure the cover glass in place. Now, close the dermis with a 5-0 suture and a needle holder. Then, place the mouse in a new cage with clean bedding, with monitoring until it is fully recovered.

To obtain combined multiphoton and confocal microscopy images through the window chamber, first, inject the appropriate fluorescent vasculature neutrophil labeling solutions into the mouse by tail vein. Clamp the ends of the window chamber with alligator clips to affix the mouse to custom-made stage. Then, place the imaging stage onto the combined multiphoton and confocal microscope.

When the mouse is secure, turn on the two-photon laser and the confocal system, and click Start System to launch the image acquisition software. To set up the imaging channels, under the Acquisition tab, click Smart Setup, and select the lasers for imaging. Choose Best signal, and click Apply.

Manually add the second harmonic generation image acquisition channel by turning on the two-photon laser in the Laser window, and setting the appropriate acquisition wavelengths for second harmonic generation. Under the Locate tab, click Oculars online, and locate the region of interest through the microscope by visual inspection. Click Oculars offline, and open the Acquisition tab.

In the Channels window, select the FITC Track, and click on Live to preview the FITC-Dextran image with a 5x objective. Adjust the focus as necessary. Then, click Stop under the Acquisition tab to stop the preview.

Now, manually select the 20x water objective and view the FITC-Dextran image again, adjusting the master Gain and Pinhole under the Channels window to achieve the best signal and contrast. When an optimal image has been obtained for all three channels, select all of the channels in the Channels window, and click Snap under the Acquisition tab to acquire a 2D snapshot. Next, to acquire a 2D time-lapse image, select the Time Series to open the Time Series window, and enter zero for the intervals and 40 for the total number of scans to obtain 40 images continuously without a time interval.

To analyze the images, open the appropriate image analysis software, and open an image file. To generate object tracks using the time-lapse images, in the Surpass view, open the image to be analyzed, and click on the icon with an image of orange dots to open the new Spots window. After setting Tracking parameters, click the Fill gaps with all detected objects box to start the tracking.

And click the blue arrow to go to the next step. Under the Filter Type, select the Track Duration filter, and click on the green arrow to finish the tracking. Then, check the generated tracks, going back to the previous step as necessary.

To measure the cellular movement, defined by the Object tracks in the time-lapse images, under the Edit Tracks window, select the Tracks box, and select a track that corresponds to the movement of the cell of interest. The selected track will be highlighted in yellow in the time series image. Finally, under the Statistics tab, click on the icon with an image of a graph to open the Statistics window.

Then, under Selection, click on the down arrow to generate the Track Speed Mean. Vasculature within the bone marrow can be visualized by intravascular dyes, such as FITC-Dextran. Since the contrast is dependent on the injection of the dye, the acquired image represents the functional vasculature where there is sufficient blood flow.

These images of the same area of the bone marrow at different time points demonstrate the presence of neutrophils in both the intravascular and extravascular spaces over a period of several days. The imaging of this cortical bone, mainly composed of collagen fibers, was performed by second harmonic generation, further illustrating the infiltration of the vascular tissue by neutrophils. These neutrophils were tracked in the bone marrow vascular niche with no additional time interval between each scan, allowing for the accurate tracking of their movement.

The speed of the tracks generated by the image analysis software can then be measured to analyze the velocity of each cell. Following this procedure, other methods, such as using ex vivo label cells or transgenic reporter mice, can be used to evaluate other biological processes in situ, such as the influence of immune cells on tumor progression and metastasis. After watching this video, you should have a good understanding of how to install window chamber on the murine femoral bone for tracking neutrophils through the bone marrow vascular niche and collagen structures.