b. Turn the power switch and key to on position for blue laser.

Transcription

1 OLYMPUS FLUOVIEW 300 CONFOCAL MICOSCOPE OPERATION PROCEDURE 1. Turn ON microscope in this order: 1) Turn on mercury lamp (Note: once the mercury lamp is turned off, DO NOT turn it back on for at least 10 minutes. Otherwise the service life of the mercury burner will be shortened) 2) Turn on lasers in this order: (laser need to warm up for about 15 minutes before imaging) a. Turn on the key to (on) position for green and red laser. (two small black boxes) b. Turn the power switch and key to on position for blue laser. 3) Turn on Olympus BX-UCB control box 4) Turn on Olympus FV5-PSU power supply unit 5) Turn on Olympus LG-PS2 light source 6) Turn on computer if not already on. Password = fluoview 7) Start software by double clicking on the Fluoview Microscope Icon on the desktop. 2. Lower the sample stage by pressing the TOP green button on either side of the front of the microscope.

2 3. Load sample on the stage and raise the stage by pressing the TOP green button on either side of the front of the microscope. 4. Open the Microscope Control by clicking on the microscope configuration icon 5. Find sample by using the transmitted light 1) Make sure the light path selector knob is in BI postion

3 2) Make sure the Shutter is closed 3) Turn on the transmitted light by click lamp icon 4) Click none or DIC in the Mirror Unit wheel 5) Select the objective lens in the Nosepiece wheel (normally start from the 10X or 20X) 6) Use the XY stage control to move sample and find the imaging area in the sample 7) Use focus knob on either side of the microscope to focus the sample 6. Florescence imaging 1) Turn off the transmitted light by click on lamp icon 2) Choose the appropriate filter for you sample (eg. CY3 for TRITC, FITC for FITC, etc.) 3) Open the Shutter 4) You should see your sample fluorescing. If not, the make sure you have the correct filter in and make sure you have focused on the sample.if you still do not see anything, then check your fluorescent labeling protocol 7. Using immersion Oil for the observation and scanning 1) Only 40X and 60X objective lens can using immersion oil for observation and scanning! 2) Switch objective lens to 40X or 60X, and focus sample 3) Lower the sample stage 4) Put a drop of immersion oil on the center of your sample slid coverslip 5) Raise the stage and focus sample again 6) Do not put oil on a non-oil objective lens (4X, 10X and 20X), Do not switch back to the non-oil objective lens after add oil on the sample slid coverslip! 8. Lasing scanning 1) Pull out the light selector knob to LSM position 2) Set up lasers: a. Click on the laser icon to open the laser window b. Click the box that says Rdy to turn off the laser you are not using Note: we have three lasers: Blue, Green, and Red. Do not change laser intensity! 3) Select the appropriate dye a. Click the yellow ball icon b. Find your dye from the dye list

4 c. Click and hold the selected dye and drag it into the appropriate channel. Make sure the correct channel box is checked d. Select the Detection mode slider: Push the slider all the way IN for dyes with wavelength shorter than 570nm (eg. FITC); MIDDLE position for dual-labeled (eg. FITC and TRITC); OUT postion for dyes with a wavelength greater than 570nm (eg. TRITC) 4) Set up image acquisition setting a. Scan size can normally be 800X600 for normal images and 1024X1024 for publications, and you should have the appropriate aperture in place b. Choose the san speed (normally fast for over viewing sample, slow for imaging) c. Choose the Zoom d. Click on XY repeat button to view the sample scanning image on the computer screen

5 e. Adjust image brightness by adjusting PMT (normally Gain is 1, Offset is 0) 5) Setting Z-stack scanning a. Click on XY repeat button and get the scanning image on the computer screen b. Click Set zero on the Z stage subpanel c. Use the up arrow to find the top of your sample (or as far up as you want to image), then click Set on the top Z box d. Use the down arrow to find the bottom of your sample (or as far down as you want to image), then click Set on the bottom Z box e. Set the step size according to your sample. As a general rule, the larger the step size, the quicker the scan, but less information you get, vice versa. Normally slices for a normal resolution scan. f. Click stop scan g. Change your scanning speed to Med or Slow

6 h. Click on xyz to change scan option to xyz (next to XY repeat), then click XYZ on the top middle of the panel i. Z-stack scan will begin. When the scan is completed, a box will pop up on the top left corner j. Click on Z icon and click on the bottom Z of the drop down menu. All the stacks will be compiled to form a 3-D image of your sample. k. Save the image and Click series done. Repeat from step 5 to 8 for additional imaging. 9. When finished acquiring images: 1) Save all the images you need 2) Lower the stage 3) Collect sample slide 4) Use lens cleaner and lens paper clean the objective lens if you have used immersion oil on the lens 5) Raise the stage 6) Close the software 8) Turn off the Olympus LG-PS2 light source 9) Turn off Olympus FV5-PSU power supply unit 10) Turn off Olympus BX-UCB control box 11) Turn off the power switch and key on the blue laser box 12) Turn off the green and red laser box 13) Turn off the mercury lamp