Transcript
MIF ZEISS FLUORESCENT USER PROTOCOL - FLUORESCENT START-UP
Turn on the X-Cite Fluorescent Light source.
ICc 1 – Camera #2
MRm – Camera #1
Turn on the power supply to the left of the microscope. Turn on the microscope. Allow the microscope to start up fully. Turn on the computer. Choose the Zeiss icon. Open the Axiovision Rel. 4.8 software. SETTING UP THE WORK POSITION FOR YOUR SAMPLE
Load your slide onto the stage by lowering the stage using the buttons on the lower right of the microscope. The Set Work Position screen should be visible on the touch screen attached to the microscope. Choose the 20x objective on the Menu bar. Select FL Shutter Open. Make sure the TL Shutter is closed. Select your fluorescent channel (ex: Alexa 488). Using the coarse focus knob, focus your sample. Once the image is in focus, select “Set Work Position” on the touch screen attached to the computer. The microscope will use this information to return the stage to this Z position when you change slides. FLUORESCENT IMAGING
To take fluorescent images, use the Workflow Menu “Fluorescence” that runs down the left side of the computer screen. Start by selecting the 20x objective. Select the AxioCam MR Rev3 camera. You can either click the 1st AxioCam MR Rev3 button on the workflow, or you can manually change between cameras by using the black buttons on the upper right of the microscope, directly under the cameras. The MR Rev3 is Camera #1.
Zeiss Fluorescent User Protocol – Fluorescent Imaging SOP (05-12)
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Selecting the 2nd AxioCam MR Rev3 button on the workflow will open the AxioCam MR Rev3 dialog box. Click Light to AxioCam MRM. This will direct light to the camera. Click the Live button. This will show a live picture of your image on the screen. Click Measure in the AxioCam MR Rev3 dialog box. The computer selects an optimal exposure time. To check to see if any pixels in your image are saturated, turn on the Over Exposure Indicator, located at the bottom of the Live image window. Over saturated pixels will appear orange. Decrease the exposure time until all of the over-saturated orange pixels are gone.
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MULTIDIMENSIONAL ACQUISITION – CHANNEL SELECTION
Multidimensional acquisition allows you to take multichannel fluorescent images both in 2 and 3 dimensions. Select the Multidimensional Acquisition tab in the Fluorescence Workflow. To define the channels you want to use, select the C tab, and right-click on the channels you want to turn off. When it is turned off, the channel will become grey and have an X through it. You need to set the exposure for each channel you wish to use. Select the channel in the Channel Definition panel, and select Measure. A new Exposure box will appear with your image. Select Measure to get the exposure time for that channel. Select OK. Do this for each channel you are using When you have finished adjustments, click Start.
MULTIDIMENSIONAL STACKS
making
ACQUISITION
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Z-
To take a Z-stack image, select the Z tab, and determine your slice thickness.. When you have set up your experiment, return to the Experiment tab and select Start. SNAPPING AND SAVING YOUR IMAGE
When you have finished making adjustments to your image, snap the image by selecting Snap from the Fluorescence Workflow menu. Save your image to the desktop, or to your flash drive. SHUT DOWN
Close all open dialog boxes on screen.
Zeiss Fluorescent User Protocol – Fluorescent Imaging SOP (05-12)
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Shut down the computer. Turn off the microscope. Turn off the power supply. Turn off the X-Cite Fluorescent Light source. Cover the microscope.
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