Leukocyte recruitment plays a significant role in host defense against infection, and one unchecked can contribute to inflammatory and autoimmune diseases. Here leukocyte recruitment and migration is visualized by first injecting quantum dots into a mouse to fluorescently labeled the blood vessel lumen, then injecting bacteria into the footed subcutaneously. The mouse is then transferred to a prewarm stage where its paw is secured to the cover glass of the imaging chamber.
Images of in vivo neutrophil recruitment and migration are then acquired using two photon microscopy and the various steps in leukocyte recruitment from rolling and adhesion to diapedesis and chemotaxis are quantitatively analyzed. Hi, I'm Wan from Dr.Mark Miller's in the Department of Pathology and Immunology at Washington University in St.Louis. Today we'll show you a procedure for non-invasive two photon imaging of leukocyte, homing and migration in vavo.
My name is Jeremiah McDowell. I'm also from the Mark Miller lab. We use this procedure in order to study monocyte and neutrophil recruitment dynamics during inflammation.
So let's get started In this protocol, adult lys, M-E-G-F-P, mice are used in these mice. Neutrophils and macrophages are EGFP labeled. Begin by diluting 15 microliters of non-targeted quantum dots into 100 microliters of PBS inject into a mouse intravenously for later visualization of blood vessels.
During imaging about 10 minutes after injecting the quantum dots, place the mouse in an isof fluorine anesthesia chamber and set the isof fluorine to 3%and the oxygen flow rate to approximately one liter per minute. Once the mouse has been anesthetized, perform a toe pinch to check adequate depth of anesthesia. Apply pure lube veterinary ointment to the mouse's eyes to prevent them from drying out.
Place the mouse on the surgery stage with an anesthesia nose cone and swap the footed of one hind paw with 70%ethanol. Adjust the isof fluorine to between 1.5 and 2%to maintain anesthesia. Monitor the breathing rate of the mouse throughout the imaging session to ensure an adequate plane of anesthesia Using curved forceps, grasp the needle of an insulin syringe near the bevel and bend it 45 to 90 degrees.
This will facilitate injection and improve the precision of the injection depth pipette. Point five microliters of appropriately diluted bacteria into the lid of a micro huge tube. Then load the syringe with curved forceps.
Hold the middle toe of the rear left paw and insert the needle. Gently bevel up through the skin in the middle of the distal phx. Inject slowly hold the needle in place for an additional five seconds to prevent material back flush.
The imaging platform consists of an aluminum plate with a circular hole cut through the center. A large cover glass is glued to the bottom of the plate and a rubber O-ring is glued inside the hole. To create a fluid tight recessed chamber to accommodate the rear paw, warm the aluminum base plate to 36 degrees Celsius by attaching two heating elements to the top of the plate.
Please the mouse on the prewarm imaging stage and warming pad. Maintain the mouse's core body temperature using the 36 degrees Celsius warming pad secured adjacent to the heated imaging stage base plate. Apply a thin film of super glue, then secure the paw to the cover glass of the imaging chamber.
Wash the paw twice to remove any floating bits of glue. Then fill the chamber with fresh prewarm PBS. The two photon microscope used in this protocol is a custom built dual laser video rate system built around an electrophysiological upright microscope stand.
The system is equipped with two ty sapphire lasers for head-on by ALK I PTs for simultaneous three and four channel acquisitions and a 20 x water immersion objective. Tune the chameleon XR T Sapphire laser to 890 nanometers by using the wavelength adjustment knob and press select next place 480 nanometer and 560 nanometer dichroic mirrors in the detector head for three channel separation. Carefully lower the objective into the PBS and focus on a vessel of the toe with the image acquisition speed.
Operating at video rate 30 frames per second and using the second harmonic signal emanating from collagen as a tissue landmark. Quickly survey the tissue in real time to locate a blood vessel of interest in this case. One with em.
EGFP neutrophils present. Adjust the gain on the PMTs to optimize color separation and to minimize the laser power required to produce sufficient signal over background. Be careful not to saturate the image or photo damage the cells.
Once a region of interest is located, set two different temporal resolutions. Video rate imaging for capturing fast moving cells in a single plane and time-lapse imaging for tracking cells migrating in the extravascular tissue in 3D for imaging cell rolling and adhesion within blood vessels. Acquire 30 frames per second within a single Z plane for documenting cell extrication and migration in the tissue parenchyma input parameters for 3D time lapse imaging.
A typical acquisition protocol will consist of averaging 15 frames for each Z step and acquiring 21 sequential 2.5 micrometer steps for a volume of 200 by 225 by 50 micrometers at 32nd intervals. Mice can be imaged for up to four hours without compromising the animal's viability. Those sessions of less than two hours are recommended if longitudinal studies are to be performed.
Following the imaging procedure, gently released the rear paw with a small amount of acetone. Then place the mouse back in its cage for recovery. Wait 24 to 48 hours between sessions to minimize the risk of anesthesia, overdose or dehydration.
Once the data files are stored on the lab server, emirs or velocity software can be used to perform multi-dimensional rendering and cell tracking.Here. Time-lapse imaging shows neutrophils in green rolling along the endothelium of blood vessels about 10 minutes after infection with listeria monocytogenes shown in red. Collagen fibers in the connective tissue appear in blue in this movie.
Typical neutrophil recruitment from circulation and migration through inflamed tissue is shown. We've just shown you how to noninvasively image, leukocyte migration, and homing. And the mouse foot pad when performing this procedure is important to remember that the injection depth in the toe is not too deep.
So that's all for now. Thanks for watching and good luck with your experiments.
