Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

The overall goal of this procedure is to demonstrate the preparation of biological specimens for sectioning and imaging under the knife edge scanning microscope, or KESM and the visualization of the resulting image stack data. This is accomplished by first fixing, staining, and embedding the tissue to prepare it for imaging under the KESM. The second step of the procedure is to configure the KESM and conduct sectioning and imaging using the automated control software.

The resulting images are then de-noise registered and cropped. The final step is to load and explore the data sets in a web-based atlas and 3D visualization software. Ultimately, results can be obtained that show whole brain level, neuronal and vascular organization at sub micrometer resolution.

The main advantage of this technique over existing Mesos, such as the serial block base scanning electromicroscopy, is that it can image much larger volumes, albeit at a lower resolution. This method can help answer key questions in the field of connectomics and neuroscience in general. This method can provide insight into the structural organization of the brain, and it also can be applied to other organ systems such as the lung and kidney.

To begin this procedure, the dissected mouse brain is placed in a glass jar containing Golgi Cox Solution. Place the glass jar in a light tight box at room temperature for 10 to 16 weeks. A separate fixation step is not necessary since the Golgi Cox solution works as a fixative.

After 10 to 16 weeks, rinse the brain in deionized water overnight in the dark on the following day, immerse the rinsed brain in 5%ammonium hydroxide solution in deionized water for seven to 10 days in the dark at room temperature. After seven to 10 days, rinse the brain again in deionized water at room temperature for four hours. Then dehydrate the brain through a graded series of ethyl alcohols, leaving it in each solution for 24 hours, 50%ethanol and 70%ethanol at four degrees Celsius and 85%ethanol.

Three changes of 95%ethanol and four to five changes of 100%ethanol at room temperature. Next, put the dehydrated brain in acetone and perform three to four changes each for one day. After this, put the brain overnight in the refrigerator in each of these aite acetone mixtures with an aite to acetone ratio of one to two, one to one, and two to one.

Finally put the brain in 100%aite in the refrigerator, perform three changes each time overnight. The treated brain is then embedded in 100%aite and heated to 60 degrees Celsius for three days. Finally, the cured specimen block is mounted on the metal specimen ring, using it epoxy and the sides of the block trimmed as necessary to begin this procedure.

Confirm that the mouse is deeply anesthetized and non-responsive to a toe pinch. Then perfused the mouse trans cordially using room temperature phosphate buffered saline, followed by cold 4%phosphate buffered para formaldehyde. Next, perfuse the mouse with a solution of 0.1%thi and dye in deionized water.

After that, place the body in a four degree Celsius refrigerator for 24 hours. After 24 hours, remove the brain from the calver and place in a fresh solution of 0.1%Thionine. Leave the brain at four degrees Celsius for seven days.

After seven days, detain and dehydrate the brain through a graded series of ethyl alcohols. Over a time period of six weeks, three to five days in 50%ethanol, seven to 10 days in 70%ethanol about two weeks each in 85 and 95%ethanol, and the rest of the time in 100%ethanol. After three changes of acetone at room temperature, the brain is embedded in erudite plastic in a 60 degree Celsius oven.

To begin the procedure for staining the vascular network with India ink, confirm by a lack of toe withdrawal reflex that the mouse is deeply anesthetized. Then perfuse the mouse trans cordially using room temperature phosphate buffered saline, followed by room temperature, 10%neutral buffered formin. Whole body perfusion with saline and fixative is necessary to clear the blood from the cardiovascular system and to fix the tissues.

Next, perfu the mouse with 3.0 ccs of Undiluted India ink perfusion. With India ink is necessary to completely fill the vasculature of the cardiovascular system. After its removal from the mouse, the resulting brain is dehydrated through a series of graded ethyl alcohols and then embedded in erudite plastic.

Following the protocol shown earlier to set up the KESM for imaging, turn on the camera, illuminator and stage, raise the knife and objective. Next, install the specimen ring. Then lower the objective and knife.

Measure specimen dimensions. Then create a configuration file for the KESM stage controller. Two application, raise the knife and the objective of the KESM prior to initializing the stage.

Start the KESM stage controller two and initialize the stage. Then lower the knife and objective and turn on the pump. Focus the objective and camera by turning the focusing knob on the optical train and adjusting the camera's field of view relative to the knife edge.

Click, step-by-step to check the first few steps of the imaging and sectioning. Then click go. To initiate imaging and sectioning for image processing.

Run the KESM stack processor to remove lighting artifact and normalize the background intensity in all images. Repeat for all data subfolders. To visualize data using the KESM brain atlas, go to this website and select the appropriate atlas to go to a different depth.

Select from the pull down menu, how deep to go at each step, and then click on either plus to increase or minus to decrease Z depth. Other options include zooming in or navigating using the KESM brain atlas. Use the pull down menu to adjust the overlay number and the overlay interval to visualize data using VIS lab.

Launch the application, create a new project. The project should include compose 3D from 2D files set world matrix arithmetic one and view 3D connected. In that order, appropriate parameters should be set as shown in view 3D while pressing the right mouse button.

Move the mouse around to adjust the threshold regions can be cropped. The orientation changed, the illumination changed, or the background turned on or off. Representative KESM results are illustrated by the following Video clips shown first are volume visualizations of KESM, whole Brain India ink data stacks for the vascular data set, beginning with a closeup of the vascular data with a width of about 100 microns.

This second clip of KESM, whole Brain India ink data shows three standard views of the whole mouse brain vasculature, subs sampled from high resolution data with a width of about 10 millimeters. The sagittal view, the coronal view, and the horizontal view, as well as a fly through of the coronal section.KESM. Whole brain mouse Golgi data is illustrated in this video clip that shows a fly through along the three different sectioning planes, starting with the block view, followed by the sagittal view, the coronal view, and the horizontal view.

Next details of the KESM whole brain mouse GoGy data are shown. This first video clip is of the cerebellum and purkinje cells. This second clip shows the cortex and parametal cells.

Finally, KESM. Whole brain nle data is illustrated in these two video clips. Shown first is a closeup of the NLE data.

This second clip shows three standard views of the whole mouse brain Nle data subs sampled from high resolution data. A cross section is also shown for a clear view of the internal structures After this development. This technique paved the way for researchers in the field of neuroscience to explore neuronal and vascular networks in the mouse brain.

After watching this video, you should have a good understanding of specimen preparation, imaging, and data analysis for the knife edge scanning microscope.