Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Our protocol allows researchers to analyze the process of arteriogenesis in vivo by tracking immune cell adherence and extravasation in growing collateral arteries in real time. The multiphoton microscopy technique allows for the visualization of cell dynamics in the deep tissue of living mouse models with low phototoxicity and with high spatiotemporal resolution. The microscope procedure will be demonstrated by Dominic van den Heuvel, a technical assistant from the multiphoton imaging platform from the laboratory of Dr.Helen Ishikawa-Ankerhold.

I will demonstrate the surgical procedure. To begin, place the anesthetized mouse in the supine position. Then place two pieces of molded clay under the upper hindlimb of the mouse to ensure a plane position of the adductor muscle.

Place the mouse under the stereo microscope. After removing the hair from both legs, disinfect and cut the skin in a circle around the scar of the earlier femoral artery ligation of the right hindlimb. Remove the skin and fat layer from the top of the leg and pull aside the remaining skin using sutures to create a pocket around the adductor muscle with the collateral vessels.

Next, remove the subcutaneous fat to get a clear vision of the adductor muscle, the profunda artery and vein, as well as the collateral vessels. Remove the superficial muscle layer on top of the collateral vessels using fine forceps. Fill the prepared pocket with saline to prevent the tissue from drying and continue the same procedure for the sham operated left hindlimb.

Before imaging, add ultrasonic gel in both pockets which prevents drying and serves as an immersion medium for optical coupling with the objective. Turn on the key of the titanium sapphire laser box, the electronic interfaces box, the heating unit of the incubator chamber, the fluorescent lamp, and the computer. Launch the acquisition software.

Set the wavelength to 800 nanometers and open the microscope shutter. In the measurement wizard dialog, choose the instrument mode as single beam and measurement mode as 3D scan timelapse. Then transfer the anesthetized mouse into the pre-warmed incubation chamber of the microscope and position the area with the ultrasonic gel directly in contact with the objective front lens.

Open the epifluorescence microscope shutter. Then choose an adequate filter or dichroic setting for FTSE visualization. Define the area of interest by following the blood flow under epifluorescence illumination.

After bringing the area of interest into the center field of view, close the epifluorescence microscope shutter. In the XY scanner dialog, set the required parameters for image size, pixel, frequency, line average. Adjust the laser power and set the photo multiplier gain for the channels green, red, and blue.

Select an adequate objective for intravital imaging and drift correction settings. Define the image stack range by starting the preview acquisition mode by pressing the red button in the measurement wizard dialog window. To obtain good images, define the area and structure of interest by focusing.

While observing the screen, change focus by moving the objective until the image disappears and set it as zero. Set the objective position as the last position in the axial direction. Then change focus in the opposite direction by moving the objective down until the image disappears from the screen again.

Click the stop button on the top left corner of the measurement wizard dialog and set the step size to two micrometers. Choose the python axis as the first axis device in the measurement wizard dialog and select do not activate the auto-save mode. Then check the auto-save box only on the time axis.

Next, press the Python icon in the available devices window to open the Python dialog window. In the Python dialog axis settings, enter in from and to sections, then enter the number of steps. Go to the XYZ stage Z dialog window and enlarge the scan range by 200 micrometers on both ends.

To do so, enter 200 micrometers in start and enter minus 240 micrometers in end and the range will automatically be set as minus 440 micrometers. Open the VivoFollow front-end dialog. Start image acquisition by pressing the green arrowhead in the top left corner of the measurement wizard dialog.

If live drift correction is used, look for a dialog for the drift correction configuration to appear. Then set the acquisition channel used as in a mobile landmark reference. Enter the maximal correction offset in micrometers added to the first and last Z positions, and click OK.Monitor the current drift offset in X, Y, and Z in real time during the image acquisition in the vivo flow front-end dialog window and stop imaging acquisition after 35 minutes when another cycle of anesthesia re-injection is required.

Inject half a dose of the MMF mix and restart imaging acquisition until the experiment is finished. This tool allows long-term image acquisition, enables high-quality data collection, and is suitable for tracking the cells for speed measurements. As shown without drift correction, the region of interest progressively drifts away from the recording view, impacting the ability to track cells for speed analysis.

However, stable movies can be recorded with the drift correction software and more cells can be tracked over a long period. The drift correction software can also provide a visualization of the X, Y, and Z offsets over time that was system corrected. Multiphoton microscopy offers a high spatiotemporal resolution for leukocyte tracking wherein cell migration steps and speed can be tracked and monitored.

By preparing the collateral vessels for multiphoton imaging, it is essential to avoid damage to the collateral arteries. Before imaging, it is important to safely identify the correct collateral artery. With this new procedure of intravital multiphoton imaging of the collateral arteries, it is possible to analyze adherence and extravasation of all blood cells in vivo by changing the antibody label.