1. Miriam Israelowitz 1 and Dr. David L. Wilson 2 1 Department of Physics, Case Western Reserve University, Cleveland OH, 2 Deparment of Biomedical Engineering, Case Western Reserve University, Cleveland OH Abstract Fluorescent microscopy has become the method of choice when imaging stem cell growth. One of the fastest growing science fields today, stem cell imaging research exploits the ability of fluorescence to image the tiny cell amounts. The nature of such imaging increases the sensitivity to extraneous fluorescence and it becomes very important to reduce excess signal as much as possible. Experimental methods can effectively erase external noise sources such as compounds prone to creating excess fluorescence, however it cannot compensate for the intrinsic tissue specific fluorescence present in all organic matter. This project attempts to address autofluorescent issues by measuring the intrinsic fluorescence for each specific organ tissue with in a range of light wavelengths. These calibration curves can be recording into a database which can be used in signal processing in combination with experimental techniques. The method of imaging that these calibration curves will be implemented is a novel new method called Cryo-imaging. The Cryo-imaging system cryogenically freezes mice that have been injected with genetically modified stem cells. It then systematically sections and images the frozen block faces. Two cameras with optical filters are mounted on a robotic arm to capture the emission light. This experiment uses a Nuance multi-spectral camera which has an adjustable emission wavelength capture filter to measure the spectrum of autofluorescence. The autofluorescent spectrum collected showed that the stomach, liver, and brain produce the most autofluorescence while the intensity from the heart and lung were almost negligible. Characterizing Autofluorescence in Rodent Tissue Using the Cryo-Imaging Method Methods The autofluorescent spectrums were captured through a system consisting of a microscope, optical filter cube, and Nuance Multi- Spectral Imaging camera. Results Figure 3: Long pass filters at the excitation side and emission side of the filter cube limit the range of wavelengths that may pass through to the camera. The range of wavelengths allowed through the excitation filter is shown as blue colored area. Only the range above 488nm is allowed. The range of wavelengths allowed through the green emission side filter is shown as the area colored green. The cutoff wavelength is 540nm. The filters ensure that only fluorescent light will enter the camera. The autofluorescent spectrums were taken from 500nm to 950nm in increments of 10nm. The imaging time for each wavelength was 20ms. Figure 1: Schematic of Imaging system. A fluorescent 100V light supply is directed through a coaxial light cable to a blue wavelength excitation filter. The filter allows light ~488nm to enter the optical filter cube. A dichroic mirror directs light to the sample and allows only light above 488nm to pass through to the emission side. The emission side filter is a long pass filter which allows only light above 540nm to pass through to ensure only the fluorescently produced light is recorded. The Nuance Multi-spectral Imaging Camera is equipped with a filter which can Figure 4: Experimentally found spectrum of the autofluorescence of various organs. The organ spectrums are compared to the spectrum of a reference slide which emulated the fluorescence of a GFP cell. The intensity of the signal is dependent on the duration of the camera capture time. It can be seen that the stomach, as expected, has the highest amount of autofluorescence present. The liver and the brain are the next two organ tissues to emit a high autofluorescent spectrum. Conclusion The measurements of the autofluorescent spectrums showed that the stomach had the highest intensity of fluorescence. This is because of the high concentration of fluorescent molecules that pass through the digestive system, such as chlorophyll. Placing the mouse on a chlorophyll-free diet helps reduce the amount of extraneous molecules, but it cannot completely eliminate the autofluorescence. Other tissues that have high fluorescent intensities are connective tissues. The heart and the lung have such a low spectrum of intensities and it can be assumed that the autofluorescence is negligible in these tissues. The stomach has a peak value at 580±10nm. Other studies have shown the peak intensity of the stomach as 560nm. The measured value, although out of range of comparative studies, is within a reasonable distance. The spectrum of autofluorescence can be used in further contrast enhancement techniques. In the Cryo-Imaging machine set up, the camera system is positioned above a mouse-sized Cryo-stat chamber kept at -22°F. The chamber contains a motorized platform which can be controlled through a panel or a computer. The platform is positioned under a knife and can be moved, raised, and lowered to cut slices that are micrometers thin. The entire imaging system is mounted on an xyz robotic arm. The arm is able to slide the microscope and camera system over the Cryo-stat chamber and lower the apparatus in. change its allowed wavelength in increments of 10nm. The camera is powered by a 5V supply. The images taken are transferred to the Nuance software program for analysis. Figure 2: Scope of the Nuance Imaging system. After the block face of the mouse has been exposed, the sample can be imaged in brightfield and fluorescence. The upper left hand image is the brightfield image taken in white light. The upper right hand image is the sum of the images taken through fluorescent imaging over a range of emission side wavelengths. The lower left hand image shows how the Nuance Imaging system is able to separate the distinct organ fluorescence wavelengths from the background image. The final lower right hand image shows the separated wavelengths that are fluoresced, indicated through different colors. In this image, it can be seen that the internal organ tissue fluoresce at a higher wavelength than skin tissue. Spectrum of Excitation, Emission, and Long Pass Filter Wavelengths Future Work The spectrum of autofluorescence can be used in combination with imaging techniques to give images of the mouse block faces. The true signal of the GFP cells can be found by subtracting off the intensity caused by autofluorescence. Acknowledgements I would like to thank Debashish Roy and Grant Steyer for all their help. I would especially like to thank Debashish Roy for his patience and willingness to help make the project possible.