Standard Operating Procedure For Zeiss Confocal Microsope

Transcript

Franceschi Microscopy & Imaging Center Washington State University Standard Operating Procedure for Zeiss Confocal Microsope Purpose: Proper operation of Zeiss confocal microscope Training Required? YES – Do not attempt to use this protocol until you have been trained by FMIC staff Protective Equipment; NA Turning On the Scope and Computer 1. Turn on the power to the Mercury Lamp #1. Then press the ignition button, hold down for several seconds, a yellow Lamp Ready light should turn on. 2. Turn key (Emission Off/On) #2. Fan will start and you will hear beeping. Wait for beeping to stop. 3. Turn on computer #3. Wait for computer to full turn on. Press Ctrl+Alt+Del to login with prompted. The password is “password”. 4. When computer is fill booted push the ON button on the Controller Box #4. This is the big tower next to the computer. 5. Double left click LaserSharp2000. When it says Default user on login scale down to EMC. Password is “EMC”. Wait for program to fully start. Viewing Samples on the Microscope 1. Place your slide coverslip down on the scope. To view with ocular lenses turn the dial on the right side of the scope to the “A” position. Choose the appropriate filter for you sample’s fluorescence by sliding the handle under the microscope stage. If you are not using fluorescence, turn the lamp on (left side of scope) to help you find your sample. Reflected/Transmitted Mode 1. Change filtlers so that B1 is in position 1 and OPEN is in position 2 (when you are done please return filters to storage box and replace the originals so that T1 is in position 1 and T2 is in position 2). Do not turn on the mercury lamp. 2. Select Reflected Blue. Viewing Samples on the Computer 1. Make sure that the dial on the right side of the scope is in the “C” position (Rotate the dial 2 clicks in either direction from the “A” position). Also make sure that the lever on the top back of the scope is pointing to the left, this is the ON position for the transmission detector. A. Fluorescence Franceschi Microscopy & Imaging Center Washington State University 1. On the computer select “Method” from the top of the screen. Then select “Methods…” and choose the appropriate colors for your sample’s fluorescence. B. Reflected/Transmitted 2. To activate the laser click the star symbol. Make sure the magnification selected on the computer (4x,10x,20x,40x oil, 60x oil) is the same as the objective lense on the microscope. 3. You can focus the image by adjusting the knob at the bottom of the joystick that is by the computer. The image’s color intensity can be adjusted by changing the Krypton/Argon, Iris, Gain and Offset bars. Placing a Scale Bar on your Image 1. Double click your image of interest. Right click the image and select “Annotation” 2. At the top of the screen in the command bar a small (very tiny) scale bar will appear. Left click this scale bar and a bar will appear in the bottom left corner of your image. 3. To deselect the bar click the “Deselect image” on the top of the screen. It is just right of a red X. 4. Select Edit, then Copy. Click start  Programs Accessories  Paint. The paint program will open and now you can Paste your image. To save this image select File  Save. Saving Images 1. To save an image double left click your image of interest. This will now be the only image showing. Right click the image and select “Export” and save your image in your folder. Double click the image to return to the screen with multiple images. Or To copy to paint; select window of your image click on copy icon open paint and paste image into paint (make sure select button is chosen not the letter button) Shutdown instructions are on the wall to the left of the computer monitor.