Fluorescence Microscopy techniques have a limited ability to image structures deep inside tissue, especially in highly light scattering materials such as brain tissue. Even in the case of two photon microscopy, it can be very difficult to produce high quality in vivo images of neural structures that are deeper than 500 microns from the neocortical surface. Here, the green line indicates a depth of 500 microns.
In general, neocortical layers four, five, and six are greater than 500 microns from the surface. We will show how a one millimeter glass micro prism inserted into the neocortex can relay light into and out of superficial and deep cortical layers. Using this technique in a mouse, it is possible to have a one millimeter field of view and visualize all six cortical layers simultaneously at an imaging perspective, typically found only in sliced brain tissue.
Micro prisms offer a wide field of view capable of visualizing layer five parametal cell bodies with their long apical dendrites and the complex network of blood vessels in the neocortex, all while maintaining spatial resolutions capable of resolving dendritic spines and red blood cell flow through micro capillaries. My name is Thomas Chia and I'm A graduate student in the laboratory of Professor Michael Levine at Yale University's Department of Biomedical Engineering. The protocol we will be discussing in this video will be on how to use one millimeter right angle glass micro prisms to generate fluorescence images of structures one millimeter deep In a mouse neocortex.
The Micro prisms We use are one millimeter right angle prisms made from BK seven glass purchased from the Opto Sigma Corporation. Micro prisms are handled using forceps whenever possible. The hypotenuse of the micro prism is coated with enhanced silver to provide a reflectivity of greater than 97%from 400 to 2000 nanometers.
This provides internal reflection of both the laser excitation light and the admitted fluorescence. We use a custom built two photon microscope capable of small animal imaging. The animal attached to a stereotactic device is placed on a three axis motorized stage.
The microscope is connected to a Windows PC running scan image. Software scan image is image acquisition software written in MATLAB by Polo Gudo, et al. For micro prism imaging, we collect images at a resolution of 10 24 by 10 24 or five 12 by five 12 pixels.
In addition, we typically scan at two milliseconds per line or four milliseconds per line. An important aspect of this protocol is the type of microscope objectives used in conjunction with the micro prisms. Two types of objectives are used in the experiments.
The objective on the left is a low numerical aperture air objective for wide fields of view imaging, in this case, a four x 0.28 NA Olympus objective. The objective on the right is a 40 x 0.60 high NA air objective with a glass correction collar capable of minimizing spherical aberrations induced by zero to two millimeters Of glass. In order to prepare the mouse For surgery and the insertion of the micro prism, the first step is to properly anesthetize the animal to a level suitable for surgical procedures.
First, the animal's weight is recorded based on established dosage. An appropriate volume of a ketamine and xylazine cocktail is pulled into a syringe. The anesthetic drugs are delivered through an IP injection Using a 28 gauge needle.
Once the mouse is At a plane of anesthesia suitable for surgical procedures, the majority of the hair on the animal's head is shaved off using a tremor with a number 40 blade. Then nare is applied to the remaining hairs on the head to help create a surface free of hairs that may get in the way of the micro prism. During imaging, after five Minutes, the NA is cleaned off Using a cotton tipped swab, the anesthetized animal is properly Placed into a mouse stereotactic device.
During the surgical procedures and imaging sessions, the animal is kept on a water-filled heating pad set at 36 degrees Celsius. With the animal's head properly fixed in place, one clean incision is made down the medial line of the skull. To separate the skin, a small amount of 3%Hydrogen peroxide is placed on a cotton tip swab and apply to the skull to clear away membranes Between the skull and the skin.
The hydrogen Peroxide also helps to reveal BMA a major landmark in knowing where to perform the craniotomy and insert the micro prism. Before the craniotomy is started, the ear bars and bite bar are properly adjusted to tilt the exposed skull such that it is basically level with the table. This provides a better platform for the craniotomy.
A small dental burr is connected to a Dremel tool for craniotomies. We set the speed to approximately 7, 000 to 10, 000 RPMs. The dental burr is skimmed along the skull until a square groove is established in the bone.
The sides of the square are approximately two to three millimeters in length and centered. 1.5 millimeters coddle, and one millimeter lateral rema continue thinning the bone until a small opening is made through the skull. A pair of forceps can lift the bone flap.
The dura needs to be removed before the micro prism can be inserted into the cortex. Bleeding is best controlled by letting the blood coagulate on the surface for several minutes. Afterwards, the coagulated blood is removed using a surgical sponge to help assist the alignment of the prism with the cortex.
The vertical face of the micro prism is lined up parallel to the handles on the forceps. With the prism properly held, the entire micro prism is inserted until the top of the prism is flush with the neocortical surface. Here we can see the prism resting in the neocortex.
When inserted correctly, the micro prism should remain in the proper position. Any bleeding that results is minimal and can be absorbed with a small surgical sponge. Once the prism is in place, The animal is ready for imaging with the animal.
Under a low magnification objective, one can see the laser raster scanning over the micro prism on the surface of the brain. Here are some representative examples of images taken from transgenic mice expressing yellow fluorescent protein. In layer five cortical neurons using micro prisms, one can see a collection of large parametal cell bodies in layer five, nearly one millimeter below the cortical surface.
Also seen are the apical dendrites that extend through all the superficial layers before diverging into Tufts using a high numerical aperture objective with the glass correction collar, it is possible to resolve dendritic spines in this case. On the apical dendrites of layer five parametal neurons, dendritic spines are sub-micron structures and several are identified in the image using yellow arrowheads. One can also label the cortical blood vessels with a fluorescent dye, such as fluorescein dextran using established tail vein injection techniques.
Using this technique, one can see the larger caliber vessels from deep layers extending towards the PIA and branching off to form the network of micro capillaries. This delicate network of micro capillaries in the neocortex is best appreciated using a high numerical aperture objective. In addition, it is possible to measure red blood cell velocity and flux from deep neocortical capillaries by line scanning.
The red line indicates the line scanning pattern on the capillary image through the micro prism. Since red blood cells do not take up the fluorescent dye, they will appear as dark streaks. The image on the bottom is the result of a line scan with a spatial dimension in one AEs and a temporal dimension in the other.
AEs from this one can calculate the flux and velocity of red blood cells through the capillary. After a micro prism has been used in an animal, it can be reused several times. However, it does need to be properly cleaned to remove any remaining biological material.
This can be accomplished by dipping the prism into a small volume of hydrochloric acid, followed by a dip into methanol. The clean micro prism can then be wrapped in lens paper until feature use. This concludes our protocol on using micro prisms For in vivo cortical imaging in mice.
Using micro prisms in an imaging experiment is relatively straightforward and offers many advantages when it comes to in vivo studies on the neocortex. We hope you have found this to be an interesting technique and one that you can use in your lab in the future. Thanks for watching and good luck.
