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    • CommentAuthorjuruble
    • CommentTimeJun 18th 2007 edited
     

    This protocol explains how to: use EZ-C1 software to obtain images with the confocal lasers (including z-stacks).

    Protocol revised: 6/18/2007

    Protocol written by: James Barnes and Julie Ruble

     

    After starting up the confocal microscope and using iControl to find your specimen with fluorescence, you can use the EZ-C1 software to image the sample with the confocal lasers (this yields much sharper images than regular fluorescence -- click here for an explanation of confocal microscopy). You can also use the EZ-C1 software to take a "z-stack," which is a series of images taken at fixed intervals along the z-axis of the specimen and then compiled into a movie. Z-stacks can then be rendered to get one sharp, 3-D image of the specimen.

    INITIAL CONFOCAL SETUP

    1. Open EZ-C1 software. You should see two black windows open in the program, C1 Live:1 and C1 Live:2.
    2. Set both windows to display correctly. When C1 Live:1 is selected, both the "True Color" button ( true color ) and the correct color channel button (i.e. red if your sample glows red; Fig. 1) should be depressed. When C1 Live:2 is selected, both the "Pseudo Color" button ( pseudo color ) and the correct color channel button (Fig. 1) should be depressed.

      Figure 1. Color channel buttons:
      color channel

    3. Click the "scanhead" button ( scanhead ) to direct the light to the scanhead.
    4. Unlock the lasers by clicking the "pad lock" button ( )
    5. In iControl, change the "Filter Cube" to "DIA" instead of "Confocal" (Fig.2). This is a shortcut to fix a program bug.

      Figure 2. iControl filter cube controls:
      iControl filter cube

    6. Set appropriate color gain to ~6.00 to begin (Fig. 3).

      Figure 3. Gain controls:gain


    7. Increase appropriate laser power to ~75% to begin (Fig. 4). Going too high can photo-bleach your sample.

      Figure 4. Laser power controls:laser power

    8. Click on "Live" ( live ) to begin scanning with the lasers. Based on what you see, adjust the gain and laser power. If the image is too dim, you can also open the detector size ( detector ), but the more you open the detector, the more you bleach your sample. You can increase the "Pixel Dwell" (Fig. 5), or how long the lasers scan at each point of the sample, to increase brightness and reduce noise. Once you're happy with the appearance of the image, you can either snap a single picture (by clicking "Single": single ) or set the z-stack range (see below).


    9. Figure 5. Pixel dwell controls: pixel dwell


    SET Z-STACK RANGE:

    See Figure 6 for guidance in this section.

    1. Click the circle next to "Reference" to tell the program that you're ready to set up the parameters for your z-stack.
    2. Click the red square next to "Reference" to tell the program that you're at the "origin" of your sample.
    3. Click the circle next to "Bottom" to tell the program you're ready to set the bottom point of your sample. While watching the screen, rotate the focus knob of the microscope toward you until sample is barely visible (i.e. you're at the bottom of the sample).
    4. Click the circle next to "Top" to tell the program you're ready to set the top point of your sample. While watching the screen, rotate the focus knob of the microscope away from you until sample is barely visible (i.e. you're at the top of the sample).
    5. Click the circle next to "Reference" to save your z-stack settings.
    6. Turn "Live" scan off.


    7. Figure 6. Z-stack settings:
      z-stack settings

    CROP AREA OF INTEREST (optional):

    1. Double-click the navigation cube ( navigation cube ), which opens the navigation window.
    2. Resize area of interest to fit your specimen.
    3. Snap a single picture by clicking "Single" ( single ).


    CUT OBJECT OUT OF PICTURE (optional):


    1. Select the angle tool button ( angle tool ).
    2. With single clicks, draw around the object you want to exclude and then double-click to end the polygon.
    3. Click the "Out Spots" button ( bleach spots )
    4. Snap a single picture by clicking "Single" ( single ) to ensure excluded object is not being scanned.

    *If you are having trouble with this, see slide 31 in the Nikon confocal ppt manual on the confocal computer desktop. The Bleach/Detector setting may need to be changed.


    SCAN:

    1. Set averaging preferences on "Average" tab (Fig. 6). Averaging will help reduce noise in your image by taking numerous pictures of each slice of your sample and "averaging" them. Use the "Average" tab to set how many pictures you want to take of each slice (usually set to 3).
    2. Click "Z-stack" and "Average" green squares (Fig. 7) to indicate that you want the program to do a z-stack with averaging.

      Figure 7. Options:
      z-stack options

    3. Click "Single" scan ( single ) to take a z-stack. The status bars (Fig. 8) will show the progress.

      Figure 8. Status bars:
      status bars

    POST-IMAGING PROCESSING:

    1. Play your z-stack while adjusting its final color to ensure that the changes you make are appropriate for all frames. To play your z-stack, click on the "View" tab (Fig. 9) and press play. You may need to speed it up with the slider below the play button.
    2. To adjust color, click color tab (Fig. 9) and adjust saturation and gamma.
    3. Save z-stack as an .avi file.
    4. To create a rendered image once you have your z-stack, go to Data >> Volume >> Volume Render on the top menu bar. Then click "Enable" on the "Render" tab that shows up (Fig. 10).
    5. Save the rendered image as .tif file.
    6. Close “Average” and “Navigation” windows without saving.

    7. Figure 9. Post-image processing tabs:view and color tabs

      Figure 10. Render tab:
      render enable


    • CommentAuthorkaswart
    • CommentTimeDec 15th 2010
     
    You no longer have to change the Filter Cube to "DIA" before using the lasers; simply pressing the scanhead button and then unlocking the lasers enables their use.