1 Microscope Manipulator Joe Hippensteel – Team Leader Evan Rogers – Communications Chris Webster – BSAC John Baran – BWIG Client: Robert Jeraj, PhD. Advisor: Willis Tompkins, PhD. December 6th, 2005 Abstract: There has been an insurgence of research involving Zebrafish embryos within the last few years, primarily due to the ease with which an observer can view changes in their internal physiology caused by external and internal factors. It has been proposed that a digitally controlled micromanipulator be constructed to create a fast scanning system to image and irradiate a large sample of Zebrafish embryos efficiently. It is required that the stage not exceed 6 cm in height and have a minimum stepping resolution of 200 µm. In addition, the software necessary to operate the stage and recognize the individual fish must be developed.2Fig. 1 – Micromanipulator set-up with intergrated analog monitoring equipment. §1. Problem Definition Accurate scanning and re-positioning of samples under a dissecting microscope is inefficient with the equipment currently available to the client. The current stage is too large and the imaging and positioning hardware and software are outdated. The primary goal of this project is to develop a fused digitally interfaced stage and custom imaging technique that can systematically do the following: scan a sample of zebra fish, analyze the fused images, store the positions of each zebra fish and reposition the sample to the localized positions. An example of a micromanipulator system is shown in Fig. 1. §2. Motivation Zebrafish as Early Vertebrate Model: Zebrafish embryos are becoming more popular in the scientific community as vertebrate models. Zebrafish embryos are transparent during their embryonic stage and develop ex utero (Xu). This transparency allows for observation of organ and skeletal development on the cellular level in vivo. This is preferable to the researchers, as they are able to pinpoint and monitor a specific area or multiple areas of interest. The Zebrafish genome project is nearing completion, and will afford the scientific community many opportunities for studying this vertebrate development.3The client specifically studies the inflammatory response of Zebrafish cells due to radiation exposure, and its relationship to cell apoptosis (programmed cell death). The Zebrafish is irradiated with approximately 50 keV of non-ionizing high energy photons. The mechanisms of inflammatory response of the Zebrafish may be elucidated as a result of this cutting edge technique, which would ultimately provide insight into the mechanisms of radiation poisoning in other vertebrates. Cell Apoptosis Apoptosis is the programmed destruction of cells by their own lysosomal enzymes (Campbell and Reece, 2002). The exact pathway for human apoptosis is not currently known and is an active area of research. The cell receives a signal to die from various signaling regions of the body (e.g. central nervous system, paracrine system and endocrine system) which initiate leakage of suicide proteins from the outer membrane of the mitochondria. Post mortem, the remaining pieces of the dead cell are then engulfed and digested by neighboring cells, allowing the embryo to stay free of the harmful proteins. An example of the failure of apoptosis in the morphogenesis of the human is webbed fingers or toes (Campbell and Reece, 2002). Apoptosis has become very prevalent in current cancer research, due to tumor cells’ resistance to this mechanism. §3. Client Requirements The table must move freely in the XY plane and have a step precision of at least 200 µm. In order to clear the microscope lens, it cannot exceed 6 cm in height. It must be large enough to hold a 6 cm diameter Petri dish and have a range of motion that is wide enough4to scan the entire dish. Because each Zebrafish will be irradiated, the table must withstand 50 keV of ionizing radiation without demonstrating adverse effects or retaining any radioactivity. There should be no limit on the number of uses the micromanipulator can endure. The camera used will be purchased and integrated with the microscope. It must have sufficient resolution to see the effects of the radiation on individual fish. It must also mount easily and securely on the eyepiece and should be directly connected to the operating computer for online analysis. The imaging and positioning software must be integrated, automated, and PC compatible. Image processing software will be required to localize all embryos in a sample so that they can be irradiated one at a time. All components should be interfaced with the PC using USB or Firewire technology, but a serial port connection would suffice. The image processing software should be designed to seek out the eyes of the fish and determine the orientation of their bodies. This should be accomplished using Matlab version 7.0. Once each eye is located, the positioning software should work with the stage controller to orient each individual fish under the radiation source so that it can receive radiation. Care must be taken when moving the stage so that fish are not shifted from their initial positions. The stage translation software should be programmed in Visual Basic.5Because the amount and area of radiation must remain constant, a device should be built to position the source above the sample. It must display an accurate measurement of the distance between the source and the sample of Zebrafish. The stand should be made out of a composite material and should not be attached to the microscope or stage. §4. Design Image Processing Software One of the primary components of the automated Zebrafish irradiation system is the digital detection of the Zebrafish embryos (see Fig. 2). A program had been written by Dr. Robert Pyzalski to decipher the location and orientation of the individual Zebrafish, which could theoretically be used to irradiate the individual fish in a time optimized fashion. This was accomplished on a Linux based system using C code, interfaced with an analog camera set-up. Due to the necessity of developing a similar software suite for a PC environment with digital imaging techniques, it has been determined that Matlab version 7.0 will be the appropriate programming language because of accessibility and the team’s knowledge of it (final program with graphical user interface syntax can be found in the
View Full Document
Unlocking...