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3 channel Fluorescence overlay
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| Rhodamine | FITC | DAPI | merged channels | |||||||||||||||||||||||||||||||||||||||
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| The above images are of a test slide manufactured by Molecular Probes. Individual images were captured on a cooled CCD camera as monochromatic 12 bit images. The images were opened in PhotoShop, displayed as 16 bit images by default. For each channel, the histogram of data was 'stretched' to occupy the full dynamic range before converting to an 8 bit image. Without this step, the image would become posterized. To achieve the final color images, each monochromatic image is assigned an RGB channel to occupy: Rhodamine is in the red channel; FITC is in the Green Channel; and DAPI is in the Blue channel. For the individual color images, the other channels are filled with black. To present the compound image, each fluorochrome is presented in the respective channel within one image. | ||||||||||||||||||||||||||||||||||||||||||
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More examples of 3 channel Fluorescence overlays
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| These images were created in the same manner as described above. Here, only the multi-channel image is shown. | ||||||||||||||||||||||||||||||||||||||||||
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2 channel Fluorescence overlay
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This is a 2 channel overlay of a Sensitive Fern spore (Onoclea sensibilis L.). This is from the same slide as the sample shown on the 3-D reconstruction page. Although we have the same raw material, our image is somewhat different. The 3-D data set was collected using a confocal microscope, while this image was collected using wide field fluorescence. This image seems "fuzzy" because not only does the light that is in focus make it's way to the detector, but the light above and below the focal plane is also picked up by the detection system. Although this might make Confocal seem "better" than wide field techniques, it's really just another tool that has it's own pro's and con's. | |||||||||||||||||||||||||||||||||||||||||
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| 3 channel Fluorescence & Phase overlay | ||||||||||||||||||||||||||||||||||||||||||
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| This image is a composite of four different digital images of the same field captured using different filters. Creating an overlay of this kind of data can be done in several different ways. In this case, The Phase image as displayed in all channels of an RGB image, and each fluorchrome was assigned a different spot channel. The advantages of this technique include allowing for more than 3 fluorochromes, and unlimited color selection. For example, DAB staining or Cy5 could be added to this image, with out altering the data already presented. | ||||||||||||||||||||||||||||||||||||||||||
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