Publication number: LBL-35331
After the frog has been frozen to -30 degree C, it is placed on a metal plate. Then a sheet of aluminum foil is placed under the metal plate, and folded up on all sides around the frog. Wax is poured on the metal plate, around the frog in order to stabilize it during the vivisection procedure. As soon as the wax solidifies, the plate is mounted in a cyrotome, a machine designed to make micro meter thin slices in organic material . A sharp blade is mounted at one end of the cyrotome. The cutting procedure involves the movement of the plate toward the blade for a slice and then retraction (under the blade for another slice). As the plate retracts to the extreme end, its height is increased by a precise level of set measurement. Then, it moves toward the blade for the next slice. In this experiment , the frog is raised by 50 micron for each slice. A camera and lighting apparatus are placed over the cryotome, and are focused on the frog. The cryotome's blade is then adjusted to cut at the proper angle. With the frog, the blade, leverage, the lighting and camera set correctly, the cryotome is turned on. Inside the cryotome, the metal plate slides past a stationary blade, removing a 50 micron slice of the frog with every pass. A picture is taken of the sliced surface of the frog every five slices. By the end of this process, 136 pictures are taken.
The next step in this process involves making the photograph data assessable to computer manipulation. First, a digitizing camera is used to capture the pictures for later use in the computer. After all the photographs are converted to images in the computer, it is necessary to use some image processing techniques to align and enhance the images.
In order to display an individual part of the frog in 3D space, there is a need to segment or isolate that part. The segmentation is done on the images of the 2D slices and is called a mask, which is made up of white and black pixels. The segmentation is done using a program called Segmentation Analyzer(Segal) which uses the technique of thresholding, region growing and hand drawing to establish the boundary of the region of interest within each 2D slice. With thresholding, an area of interest , make up of a range of gray values, is given. The result of thresholding is to have the pixels that fall in a particular range to be set to white while the rest are set to black. This produces a mask of black and white value. The growing method also involves a given range of gray values. In addition one or more points(pixel) are given. Any pixels in the neighborhood of the given pixels that fall in the range of the gray values will get set to white and the rest set to black. Most of the time, the growing or the thresholding method do not segment or isolate the desired object perfectly. So the hand drawing is always used to touch up the final mask.
Segal has one additional feature which helps the process of segmentation. This feature allows a few masks to be loaded at once and enables different masks to be exclusive. When a mask becomes exclusive, its region is completely ignored by any of the segmentation methods. In other words, that region can not be drawn on. This method avoids overlapping masks when segmenting objects are adjacent to each other, like bone and muscle. If one is to start segmenting the more obvious objects then the hardest objects will not be too difficult to segment later with the help of exclusive masks.
In this project, 13 masks are generated. They are used to define the skeleton, eyes, brain, kidney, heart, lung, nerve, blood vessel, stomach, large intestine, liver, small intestine, and muscles.
Volume data is formed when all the slices of data images are stacked up. Each point in that volume space is called a voxel. A program named Sunvoxel is used to convert the 3D segmented image data of the frog into a projected view that can be displayed on the workstation screen. This program is capable of direct rendering of 3D data in a semi- transparent surface. It can handle multiple substances (e.g heart, lung, etc) with transparency and color value assigned to them. Each substance is classified by its range of voxel values. With the aid of the masks, the pixels of each substance of the frog are scaled to a certain range so that they can be treated as a distinct substance. With the classification, color, and opacity set, the frog can be rotated to any direction for viewing. By changing the opacity value of the substances, one can view objects that are blocked by other objects.
The first problem involves the inability to keep the lighting constant when taking the original set of photographs at the cryotome. The need to produce pictures that are consistently lit comes from the fact that, once inside the computer, different pixel values will be assigned to each tiny piece of the picture. Since most organs contain generally one color throughout, the entire organ is expected to have fairly consistent pixel values. Consistency of pixel values aid in mask making. When taking the pictures, the external light from a window near the cytotome was not taken into consideration. The sectioning and photography process took about eight hours, and consequently, as the day progressed, the pictures became darker as the day did. The artificial light that was provided by a lamp set up did little to stop the contrast between the daylight and night pictures.
Most of the problems encountered can be avoided in the next experiment. Misalignment can be solved by setting up markers around the frog so that every photograph captures the markers. The zoom factor of the camera lens should be kept constant throughout the photographing. Since the camera is hanging, it might be advisable to lock the zoom lens to prevent it from sliding down.
Minimal inconsistent data can be achieved by avoiding jamming the machine. The blade should be tightened frequently to avoid loosening. Also foreign objects other than the frog and wax should be avoided. The Styrofoam used to hold the frog up was believed to have caused the loosening of the blade as well. It will be wise to make note of the current frame number when the machine jams. Also try to avoid removing the metal plate as it will change the slicing angle. Lastly, avoid leaning on the machine to prevent moving the machine.
During the digitizing procedure, make sure that all four light bulbs are changed when one or more bulbs go out. It will be a good idea to have a photographer set up the camera and lighting before the slicing procedure.
A better data set should be obtained with the proper light setting. This should also help segmentation. As Dr. Licht suggested a real dissection of a frog would help the segmentation. The computer mouse is not a good device to trace objects during segmentation. An alternative device like a pen and digitizing tablet might do a better job.
Page last modified: 01/09/05|
Contacts: Bill Johnston, David Robertson