This method describes a technique to study membrane remodeling during neutrophil migration in the live animal using intravital subcellular microscopy. This protocol provides a unique approach to study membrane dynamics during animal migration under physical condition, and to uninvolve the tissue microenvironment. This technique can be tailored to address membrane remodeling and cytoskeleton dynamic organ repositioning and membrane trafficking in different cell types and migratory cells.
To begin, add the green fluorescent dye to the neutrophil suspension, purified from the wild-type mouse in 1.5 milliliters of BSA coated tube. Incubate for 30 minutes at room temperature under gentle agitation in a tube rotator. Give three washes using HBSS, followed by centrifugation at 400 times G at room temperature for five minutes, and resuspending the pellet in one milliliter Hanks'Balanced Salt Solution.
Place this suspension in the BSA coated tube in a tube rotator under gentle agitation until the injection procedure. Before the injection, resuspend the cell pellet in saline solution to reach a density of 2 million to 5 million cells per 20 microliters. Transfer the anesthetized animal to a warming pad having 37 degrees Celsius to maintain its body temperature.
After testing the paw withdrawal reflex, remove the hairs from the ear using a fine trimmer to optimize the image quality. Place the animal on its side, hold the edge of the ear, and flatten it along the warming pad using the surgical tape, then fill a syringe equipped with a 33 gauge needle with the neutrophil suspension and mount it on a micromanipulator. Gently move the needle toward the ear, and slowly pierce the skin.
Once the needle is inside the skin, slowly push the piston, and deliver two to three microliters of cell suspension. After carefully repeating the injection for two to three different areas of the ear, carefully remove the surgical tape, and let the animal regain consciousness under supervision. Allow the animal to rest for one hour before imaging to let the tissue and the cells recover from the injection procedure.
Ensure that the microscope is turned on, and the stage and lenses are preheated to 37 degrees Celsius before placing the anesthetized animal on the microscope stage. Cover the hole in the stage with a glass coverslip. Move the anesthetized animal onto the stage carefully.
If imaging is planned for more than an hour, apply the ophthalmic ointment, and secure the winged infusion-based perfusion system. Place a drop of saline on the center of the coverslip, and place the neutrophils injected ear on top of it. Gently flatten the ear using a sterile cotton swab across the center of the coverslip to remove air pockets.
Secure the ear by gently pressing a wooden stick to the side of the ear closer to the animal's head, and locking it using tape. Then find an area of interest using the microscope eyepiece, and switch to multi-photon acquisition mode. Set the laser excitation wavelength to 900 to 930 nanometers to allow simultaneous acquisition of green fluorescent dye, mTomato, and collagen-I.
Then set the appropriate set of mirrors and filter combinations on the detectors to collect the emitted light. Determine the setting most appropriate to the hardware configuration. Even if migrating cells can be tracked with 30 second to one minute intervals, image subcellular events with a higher speed of at least less than ten second intervals.
In the classical intravital microscopy approach, use a 30X lens and a Galvo scanner with an image size of 512 by 512 pixels to track the cell migration and investigate the host mouse tissue, and set the Z axis displacement using the motorized stage with a step size of two micrometers to allow the imaging of a 30 micrometer deep volume every 30 seconds. In the intravital subcellular microscopy approach, use a 40X lens and a resonance scanner with three times averaging to image the highly dynamic membrane remodeling at a higher magnification and resolution. Also set Z axis displacement using a piezo with a step size of one micrometer, allowing 20 micrometer deep volume imaging every four to five seconds.
Induce a sterile laser injury to trigger the neutrophil migration by focusing a high power excitation laser on a narrow area of 20 by 20 micrometers for 10 seconds. Identify laser induced injury by its strong autofluorescent signals appearing in all the channels, and by the resulting alteration of collagen arrangement. Save the data at the end of the experimentation for further analyses.
The recipient mouse enabled visualizing structural features in the ear tissue such as blood vessels, resident cells, and hair follicles via an intravital microscopy based approach. The laser induced injuries were easily visualized due to their strong autofluorescence detected in all the channels, and by the alterations of the collagen arrangement. A complete view of the three dimensional architecture of the skin and the localization of the injected neutrophils portrays a Z-stack of the skin from the outer to inner layers IN a three dimensional volume rendering.
Time-lapse imaging showed the neutrophils sampling the ear skin and interacting with the intracellular matrix and host tissue. Using the intravital subcellular microscopy, dynamic remodeling of the plasma membrane during migration, formation of membrane protrusions at the leading edge, and the retraction of the rear of the cells are clearly visualized. The time-lapse sequences reveal the complexity of the interactions with the intracellular matrix.
The local dynamics of the plasma membrane were analyzed using an algorithm pipeline based on the identification of 100 boundary points underlying the cell surface. The changes in the local curvature and the area underlined by plasma membrane protrusions were calculated for each boundary point, and reported for each timeframe as kymographs. Both the front and the back of the cells maintain higher curvature than the side of the cells.
Negative area changes are more apparent at the back of the cells than at the leading edge, where the positive area changes are more prominent. Following imaging, tissue can be collected, fixed, and processed for staining to further evaluate target of interest and elucidate mechanisms. This procedure designed to image membrane dynamics can be tailored to address broader cell biology question in different cell types and migrating cells in their respective environments.
