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    • CommentAuthorjuruble
    • CommentTimeDec 3rd 2007 edited
     

    This protocol explains how to: build a complete image of a neuron from photos taken of multiple frames.

    Protocol revised: 12/3/2007

    Protocol written by: Amy Reid, Julie Ruble


    After imaging a neuron from the in vitro culturing experiment, you need to put each individual frame of the neuron together to "build" an image of the entire neuron.  This will be the image that we analyze.  To build the neuron:

     

    1. Copy all pictures so you don't change the originals. 

      • Right click on the experiment folder, click Copy.  Right click on a blank space, click Paste.  This should paste a new folder with the name "Copy of Experiment X."
    2. Open Photoshop and open all pictures of one of your neurons (from the "Copy" folder).

    3. Manipulate the photos to make them look good.  For this step, be reserved.  The person who does analysis can add MORE manipulation in ImagePRO to make the neuron clearer as they measure, but they CANNOT take away from what you've already done in Photoshop.  So change a little less than you think you need to.

      • For phase images:  Image >> Mode >> Grayscale (OK to discard color info) and then Image >> Adjustments >> Levels (move gray, black, and white arrows back and forth to achieve best image).  You can also change Brightness/Contrast under the Adjustments menu.  Save the improved image.

      • For fluorescent images:  Image >> Mode >> RGB Color and then Image >> Adjustments >> Levels (move gray, black, and white arrows back and forth to achieve best image -- brightest green on darkest background).  You can also change Brightness/Contrast under the Adjustments menu.  Save the improved image.
    4. Paste all frames of a neuron into a new canvas.

      • File >> New.  Make sure new canvas is 35 inches square and is set to RGB Color, then click OK.

      • Select All of a phase image, copy it, and paste it into new canvas.  It will paste directly into the center of the canvas.  Do not move it.

      • Select All of the corresponding fluorescent image, copy it, and paste it into the new canvas.  It will also paste directly into the center of the canvas.  Now the two images are right on top of each other.

      • To link the phase and fluorescent images together, highlight both layers (hold down "shift" and click each layer), right click on one of the layers and choose "Link Layers."

      • Continue pasting all corresponding phase and fluorescent images of the neuron together into the blank canvas in this fashion.
    5. Move pieces of the neuron to "build" a complete picture.  Once all the images of a neuron are pasted into the blank canvas, you must arrange them to "build" the neuron in the following way (note that a layer must be selected in order to move it):

      • Use the "eye" symbol to make layers disappear so that only the phase images are visible.

      • Set the layer on top to "Overlay" or "Screen" mode (this setting is at the top of the layers toolbox and reads "Normal" by default) and zoom in.  Move the top layer over the bottom layer and overlap it exactly (the fluorescent layers are still linked even though you can't see them).

      • Continue doing this until you have the entire neuron built in phase.  Return the mode of all layers to "Normal."

      • Make all phase layers invisible and fluorescent layers visible just to check that the fluorescent images line up (they should, since they are linked to their corresponding phase image).

      • Once the neuron is built, make all phase layers visible only and select "Merge Visible" from the arrowhead in the circle on the right of the Layers toolbox.  Do the same for the fluorescent layers.

      • Crop out the white space around your built neuron.


    6. Save the built neuron as both a phase image and a fluorescent image.

      • With only the fluorescent layers visible, go to File >> Save As and save image as Expt#SlideLetterNeuron#f.tif (be sure to save in TIF format).  Example: 6B05f would be experiment 6, slide B, the fifth neuron on the slide, the fluorescent image. 
      • Repeat for the phase images with a "p" at the end of the file name instead of an "f."

      • Open the phase and fluorescent images again and select "Flatten Image" from the arrowhead in the circle on the right of the Layers toolbox.  Click OK to "Discard Hidden Layers."  Resave each image. 

     

    • CommentAuthorkilang
    • CommentTimeDec 10th 2007
     
    Tip for matching up the overlapping images -

    Once the images are copied into their own layers in the new canvas, change the opacity of the top image to 50% (top right of the 'Layers' toolbar). This way you can see the image below and match up common defining features between the two pictures. Be sure to change the opacity back to 100% before merging the images.