A Zebrafish Holding Chamber for Use with a Micro CT Scanner
Final Design Report
May 5, 2004
Team Members:
Austin Ramme- Team Leader
Kristen Sipsma- Communicator
Mike Haggerty- BWIG
Andrew Neumann- BSAC
Client:
Jill M. Kolesar, Jamey Weichert, and Marc Longino
Advisor:
Professor Justin Williams
Abstract:
Our objective is to build an apparatus to house one to three zebrafish during a diagnostic micro CT scanning process. To guarantee the best imaging quality, the device must be composed of materials compatible with the micro CT scanner and it must also immobilize the fish. The zebrafish must remain alive for the duration of the twenty minute imaging process. The micro CT technology is relatively new and no holding devices are available on the market for zebrafish. The device will be used for an evaluation of carcinogenesis in a zebrafish model research project currently being conducted at the UW hospital. The ultimate goal of this research is to cure cancer using drugs which eliminate the blood supply to malignant tumors.
Background:
Problem Statement
A research group working out of UW hospital is performing an evaluation of carcinogenesis in a zebrafish model. The goal of this research is to cure cancer using drugs that eliminate the blood supply to malignant tumors. The current method of zebrafish imaging is conducted using techniques that remove the zebrafish from its natural aqueous environment. Our goal this semester is to design a holding chamber to house one to three zebrafish during a twenty minute micro CT imaging process. To guarantee the best image quality, the zebrafish must be immobilized and the device must be constructed of materials compatible with the micro CT scanner. After the micro CT imaging process, the zebrafish must remain alive and available for future scans during the course of the study.
Zebrafish Background Information
The zebrafish, also known as Danio rerio, is a fresh water fish named after the five horizontal stripes on its body resembling that of a zebra [6]. The zebrafish can be a maximum length of 6 cm and has a life span of two years [7]. It prefers water temperatures of 64 to 75 degrees F and a water pH level of 6.0 to 8.0. The zebrafish is a sociable fish and prefers living in groups in the upper half of aquariums [1].
An average zebrafish need 5 parts per million (ppm) of oxygen molecules present in water to survive. They may be able to survive with 3-4 ppm but this would be stressful on the fish. Anything lower then 2 ppm will cause the fish to die [9]. Fish are cold blooded; therefore, the water temperature in an aquarium can be lowered to decrease the breathing pace of the fish. Zebrafish are bony fish which have a strong pumping system consisting of the gills and the mouth. With this pumping system, zebrafish are able to pump water in their mouth and out of their gills with out moving forward or backward [5].
Zebrafish are used frequently in research because they are prolific breeders, easy to maintain, and have large, transparent embryos [8]. They have become a valuable resource in experimental and genetic analysis and are used as a model system to better understand particular processes, organs, and diseases. Because their genome is mapped out, mutations to specific genes can show information about gene and protein function and as a result many manipulations are performed on the zebrafish [14]. A homozygous diploid progeny that carries only maternal or paternal genes can be generated to study inheritance patterns. Chemically induced mutations are able to show which genes have been used in embryo development [2].
Figure 1: Zebrafish Embryo [12]
A main use for zebrafish is for carcinogenesis research. They can be injected with known cancer causing agents to promote cancer development. Zebrafish are a useful tool to study neoplasia and to develop chemotherapeutic agents [10]. Much of the research in this area involves cloning, isolation, and structural characterization of select proto-oncogene sequences. Scientists are looking at the ability of certain genes to suppress tumors. There is an ongoing project to develop very sensitive strains of zebrafish by transferring genes that change their response to chemicals [13].
CT Scanner Background Information
CT scanners have been used in medicine since the late 1970s. CT is an acronym for computed tomography; tomos is Greek for slice and graphia is Greek meaning describing. A CT scanner uses an X-ray tube and an X-ray detector that revolves around the object being studied and takes X-ray pictures of one narrow slice. For each revolution of the tube and detector, one slice is taken and then the platform and object are moved further into the machine and another slice is taken. After all the slices have been captured, a computer compiles the slices together into one three-dimensional image that doctors and scientist examine. Advances in technology have allowed doctors and scientists to take higher resolution images faster than ever before.
Figure 2: Traditional CT Scanner [4] Figure 3: Micro CT Scanner
Today scientists use micro CT scanners to image small animals that aid in research projects. Micro CT technology has only become useful in the last 3 to 4 years. General Electric’s micro CT scanner uses volumetric CT technology, which can acquire an image at a resolution of 27 µm [3]. Volumetric CT technology differs from standard CT scanners because it uses a much larger, flat panel detector that can scan an entire organ in one revolution [11]. This technology enables the scanner to quickly collect the data needed for the computer to reconstruct the 3-D image.
Current Devices
The micro CT scanner is a relatively new technology and manufacturers have yet to produce a zebrafish holding container suitable for this type of imaging. However, devices for use with rats and mice are currently available. These devices require that the test subject be anesthetized, which is not a plausible solution for the zebrafish. After the mouse or rat is anesthetized, they are placed on a support within the micro CT scanner during the imaging process. The material used for the support varies between models. Older micro CT scanners use a small piece of wood for the support, while new models use a formed piece of carbon fiber composite.
Figure 4: Wooden Animal Support Figure 5: Carbon Fiber Animal Support
The holding chamber design currently employed by the client consists simply of a piece of cardboard. The cardboard contains slits that match the general shape of the zebrafish. In this design, the zebrafish is removed from water during the micro CT scan. As a result, the zebrafish have not survived the length of the micro CT scan in the client’s device.
Figure 6: Client’s Fish Holding Device
Design Constraints
The device will be operated directly by the client in normal lab humidity, pressure, and temperature conditions. The holding chamber will be used within a micro CT scanner valued at approximately $350,000. The chamber must not leak water into the micro CT scanner. Metal, glass, dense materials, and any other materials that will leave an imaging artifact cannot be used within CT scanner due to the nature of the imaging process. The space available within the micro CT scanner is a cylinder with diameter of 4 inches and a length of 1 foot. The imaging area is 2.5 inches by 2.5 inches. The holding chamber must weigh less than 150 grams due to the 1/8 inch thick wooden support that it will rest on. The holding chamber must be either disposable or autoclavable. If an autoclavable design is chosen, then the device must last for at least two years. The holding chamber will be used for zebrafish imaging every two weeks and only one operational device is needed. The chamber must allow the zebrafish to survive a twenty minute micro CT scan. A full product design specification is available in the Appendix.
Alternate Designs:
Design 1: Falcon Tube Design
The Falcon tube design incorporates a Falcon tube and a sponge. A Falcon tube is a plastic tube that comes with a fitted top which provides a water-tight seal. A sponge would be placed inside of the Falcon tube. The sponge would have small indentations or slits cut for the placement of the zebrafish. The fish would need to be handled directly for insertion into the slits. Falcon tubes can be autoclaved, which will allow for multiple uses of each tube. However, the sponge will need to be changed between each group of fish to prevent cross contamination between groups.
Figure 7: Falcon Tube Design
A major disadvantage of this design is that the sponge may absorb so much water that there is not enough available to the fish. Testing will be necessary to determine whether the fish can survive in this situation. Another problem is that it will be difficult to insert the fish into the slits without harming them by over-handling. The number of fish that will fit into the tube is also a concern. It is desirable to scan as many fish as possible at one time, and this device will hold a maximum of two fish. This design is advantageous because it will hold the fish almost completely still since the sponge will compress around it. The force from the compression, however, is not enough to harm the fish.
Design 2: Screw and Clamp Design
A Plexiglas container similar to a rectangular microscope slide holder would be built to hold multiple zebrafish at one time. The Plexiglas container would be 65 mm square and 50 mm high. These dimensions accommodate the 2.5 inch imaging area of the micro CT scanner ensuring that none of the zebrafish will be out of the imaging area. The container would be deep enough to allow sufficient water to keep multiple zebrafish alive for 20 minute intervals. The container would also have a channel cut where each plate (also made of Plexiglas) would be held with thumb screws. The fish would each be secured using a moveable plate that has a threaded shaft on each end to allow the thumb screws to hold it securely in place within the container. Multiple plates would be used to allow the container to hold multiple fish at once. If necessary, the plates would have holes in them to allow water to flow within the container.
Figure 8: Screw and Clamp Design
This design has various advantages. It allows for multiple fish of different sizes to be scanned at once and it completely immobilizes the fish for the scan. It also allows adequate water for the fish to remain alive. Every piece of this container would be made of Plexiglas or similar material, which would not cause interference with the micro CT scanner. This design also has some disadvantages. Each group of fish is in the same container and there could be contamination issues associated with having all the fish exposed to the same water. The container would also be difficult to make water tight. With the slot for the threaded shafts and thumbscrew setup another, larger container would have to be constructed around the smaller container to ensure that no leakage would occur.
Design 3: Cuvette Design
This design utilizes the small size of a chemistry cuvette, which has dimensions 45mm by 12mm by 12mm. The zebrafish will be placed into the cuvette and a stabilization device in the cuvette will restrict the movement of the fish. One possible stabilization device is a circular straw, which corresponds to the circular nature of a fish. If a straw stabilization device is used it will have a diameter between 6mm and 12mm depending on the size of the fish being scanned. Other materials such as a hydrophobic sponge are also possibilities for immobilization. After the fish is in the cuvette it will be capped so there is no leakage of water. A universal top will need to be fabricated for the cuvette if there is not one available on the market. This design is either disposable or autoclavable. This design will also include a tray with rectangular groves that fit the cuvettes, in which as many as six cuvettes can be held in place. This tray will be a rectangle having dimensions of 79mm by 52mm.
One of the main advantages of this design is the fact that it is very inexpensive and therefore can be disposable. This design is also very space efficient and allows for as many as six fish to be scanned at once. The cuvette is also an ideal container as it supplies plenty of water for the zebrafish. This device will be very user friendly. Also, if the straw is made out of an opaque material minimal light will be available to the fish. Therefore, the fish will be more relaxed and less likely to make large movements. In contrast, the cuvette has some disadvantages. Each individual fish will have to be sized and fitted for the correct size restraint. Minimum movement between scans may be acceptable, because the computer will still be able to compile the image properly.
Figure 9: Top View Figure 10: Front View
Preliminary Experimentation:
On February 11, 2004, our design team met to perform preliminary experimentation to help with the design process. Our team obtained twelve zebrafish for experimentation. These zebrafish were smaller than the zebrafish that would be used in the actual holding chamber; however, they were the only zebrafish available. The purpose of the preliminary experimentation was to determine the preferred orientation, response to an extremely confined environment, response to physical constraints, and survivability in 3.5 mL of water. Materials used for the experiment included chemistry cuvettes, electrical tape, aerated fish tank water, zebrafish, holding containers, eye droppers, and a stopwatch.