Imaging techniques are becoming increasingly important in the study of brain function. Among them two photon laser microscopy has emerged as an extremely useful method because it allows the study of the live anta brain and with appropriate preparations. This technique allows the observation of the same cortical area chronically from minutes to months.
In this video, we show a preparation for chronic in vivo imaging of the brain using two photo microscopy. Dr.Carl Voda, a Howard Hughes Medical Institute investigator at Janelia Farm initially pioneered the cranial window preparation That we will demonstrate today. Hi And Ricardo must from the laboratory of Carlos Porter Cayo in the Department of Neurology at the University of California Los Angeles.
Today we are going to show you the procedure for creating a cranial window for chronic in vivo imaging. This preparation is very useful because it allows one to image deep inside of the brain using to photon laser microscope. So let's get it Started.
Before beginning the surgery, Mice are anesthetized with isof fluorine. 4%for induction, one point a half to 2%for surgery, using I-A-C-U-C approved procedures, it is important to use tail and or toe pinches in order to ensure the animal is fully sedated. Using a rodent trimmer, the hair from the back of the neck up to the eyes is shaved.
Following shaving, the mouse is placed on a stereotaxic frame, resting on a water recirculating blanket set At 37 degrees Celsius, the head is firmly secured using ear bars. Eye ointment is then applied in order to prevent the animal's eye from drying out. Next, dexamethasone, 0.2 milligrams per kilogram and carprofen five milligrams per kilogram are administered subcutaneously.
In order to prevent swelling of the brain and or an inflammatory response respectively. Before beginning the surgery, be sure to sterilize the operating area by wiping the skin with three alternating swabs of 70%alcohol and Betadine. All surgical instruments have been pre sterilized using a glass bead sterilizer using scissors that have been sprayed with ethanol.
The skin over the top of the skull is removed, starting with a horizontal cut along the base of the head, followed by two cuts in the rostral direction, almost reaching the eyelids. Then two oblique cuts that converge at the midline. At this point, a drop of lidocaine and epinephrine solution is applied onto the periosteum to avoid excessive bleeding or pain with a scalpel, the periosteum is retracted to the edges of the skull.
The musculature of the back of the neck is also lightly retracted. The entire exposed area of skull is then gently scraped with a scalpel to create a dry surface. This is very important because it will allow the glue to adhere better when it is later applied.
Once an imaging site has been chosen, one is ready to create the cranial window first. A circle of about four millimeters in diameter is drawn gently with a pneumatic dental drill. After a slight drilling, lidocaine epinephrine solution is applied again onto the skull surface.
The drilling is stopped when a very thin layer of bone is left, usually one nose. This stage is reached by pushing gently on the center of their craniotomy to feel how it gives way under a drop of saline and taking advantage of the bone trabecula. A spongy structure of the bone.
The craniotomy is lifted away from the skull with very thin tip forceps. The saline is important because it will help lift up the skull and prevent bleeding of the dura. Next gel foam that has been previously soaked in saline is applied to the dura matter in order to stop any small bleeding that sometimes occurs when the skull is removed.
After drying the dura matter surface and ensuring that there is no bleeding, a sterile five millimeter glass cover slip is gently laid on top of the dura matter. A drop of cyanoacrylate based glue is applied to the opposite hemisphere on the skull. With the help of a needle, the glue is applied gently all around the window with care not to put it under the glass.
Glue can now be applied in a thin layer over the entire surface of the skull. Once the glue has dried, dental acrylic is mixed and applied throughout the skull surface, covering also a small rim of the cover slip to secure it. After securing the cover slip, a small well is made around the window with dental acrylic.
A titanium bar is also embedded in the dental acrylic. This bar will later be used to attach the mouse securely onto the stage of the microscope. For imaging, it is important to ensure that the bar is level so that it is parallel with a cranial window.
Placing a piece of paper under the bar can allow the bar to remain level while the acrylic hardens, the dental acrylic is allowed to cure or harden for 10 minutes at which time the titanium bar Has been fixed in place. The animal is then placed in a warm cage Until it recovers. After recovery from anesthesia, The animal can be imaged on the same day.
This movie shows a stack of 160 Images taken at five micron intervals with our custom build two photon microscope through a cranial window placed over the somatosensory cortex of a three month old GFPM mouse. This transgenic line of mice expresses GFP in a small subset of parmal neurons. In layer five and layer two three, the image was taken with a 40 x water immersion objective with 0.8 numerical aperture using an excitation wavelength of 910 nanometers.
In the more superficial images, it is easy to recognize the dendritic arbors of the apical dendrites of the parametal cells. The apical tuft of the dendrites of each neuron converges onto a single large apical dendrite. Smaller oblique dendrites can also be seen coming off this main apical dendrite axons crossing the field of view are also evident.
This is a stack of 12 images taking every one and a half microns using the same 40 x objective at higher magnification, a superficial dendrite is shown at high resolution enough to resolve individual Dendritic spines. We have just shown you how to perform a cranial window preparation for chronic imaging of the brain using to photo microscopy. This is an extremely powerful method for the study of the brain because it allows you to study both the structure and the function of the living brain, not only in a single imagination, but also chronically through multiple imaginations over periods of months.
Another advantage of the cranial window preparation is that a large fill of view can be imaged, which makes possible to image large structures such as an entire action or the inri arbor over a spans of millimeters. When doing this procedure, it's important to remember to work in the cleanest and more sterile conditions possible and to have a very steady hand. Okay, and that's it.
Thanks for watching and good luck with your experiments.
