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    • CommentAuthorjuruble
    • CommentTimeJun 18th 2007 edited

    This protocol explains how to: use iControl to view samples (brightfield and fluorescent) with the confocal microscope.

    Protocol revised: 6/18/2007

    Protocol written by: Julie Ruble

    NOTE: An iControl screenshot is provided below for reference while reading this protocol.

    1. After starting the confocal microscope, you will use the iControl software to control the microscope as you view your sample. Immediately after opening iControl (by double-clicking the shortcut on the desktop), you will need to open the EPI Field Stop. The Field Stop determines how wide your field of view will be, and because of a program bug, it always starts very small (4 mm). To open it, click on EPI box ( EPI box ) and drag the slider to the right.

    2. If you are using brightfield microscopy, click the "Observation Method" box on the left toolbar and then click "Brightfield." To adjust the intensity of the lamp, click the lamp box ( Lamp box ) and drag the slider. Make sure the light is directed toward your eyes (by clicking the eyepiece path button: Eyepiece button ), and not toward the camera or the scanhead, or you will not be able to see your specimen.

    3. If you are using fluorescent microscopy, click the "Custom Observation" box on the left toolbar and then click either "TRITC Bi Red" (for samples that should glow red) or "FITC Bi Green" (for samples that should glow green). At this point, turn off the lamp by click the lamp toggle button ( Lamp toggle ) and close the shutter by click the shutter toggle button ( Shutter toggle ). Closing the shutter when you are not viewing your sample ensures that you aren't bleaching it. You can open the shutter again when you are ready to view the sample.

    4. To take a QCapture picture, whether fluorescent or brightfield, you will need to direct the light toward the camera by clicking the camera path button ( Camera button ).

    iControl screenshot

    • CommentAuthorkilang
    • CommentTimeNov 11th 2007 edited

    Julie's suggestion, to protect the objective: To focus, raise the stage as high as possible without touching the objective (do this while looking at the stage, not through the eyepieces). Then, as you try to focus on your specimen (now looking through the eyepieces), only lower the stage. This way you know you will not hit the objective.

    • CommentAuthorkaswart
    • CommentTimeDec 15th 2010 edited
    A couple changes:

    -You might have to re-open the EPI field stop after every use of the lasers (with EZ-C1).
    -You can also adjust the light intensity directly on the microscope: the button on the left front of the stand allows this. If you move the knob and the light intensity does not change, click on the camera button in the front left.
    -Now, to use fluorescence, simply click on the "Fluorescence" button on the left toolbar.