MIF ZEISS VIOLET CONFOCAL ZEN 2009 PROTOCOL START-UP On the Switchbox, turn both black switches to the ON position. Wait for the microscope to boot up completely (watch the screen on the side of the microscope). Turn on the computer. Log in using your WVU Credentials: *for HS account users: userid *for Mix account users: WVU-AD\userid *for WVU healthcare: WVUH\userid Open the Zen 2009 software. Choose Start System. Switchbox Go to View Show all (global) SETTING UP THE WORK POSITION FOR YOUR SAMPLE The Zen software opens to the Ocular Menu. Online: You can visualize your sample through the eyepieces Offline: You cannot see through the eyepieces You can also open and close the Fluorescent shutter. Under Ocular, choose the objective you want to use. Be aware of oil versus non-oil objectives. This window also has the buttons to visualize FITC, TRITC, DAPI, and Brightfield through the eyepieces. Load your slide onto the stage by lowering the stage using the buttons on the lower right of the microscope. Violet Zen 2009 SOP (06-14) Page 1
Multitrack Set work position should be visible on the TFT touch screen attached to the microscope. Click the On-Line button. Find sample on the 20x objective, using both the coarse focus and fine focus knobs, if needed. Once the sample is in focus, press Set Work Position on the microscope touch screen. All of the objectives are parfocal, so you can now use the stage position buttons on the lower right to lower and raise the stage to the focal plane you have designated. TURNING ON THE LASERS Go to Setup Manager Laser menu Turn on each laser open the Laser Properties Adjust the laser output for the Argon to 40%. FLUORESCENT PROBE CONFIGURATION The Imaging Setup Menu works with the Configuration Selection drop down menu, located directly under the Acquisition menu button. The Configuration Selection Menu allows you to choose the correct filter sets and laser settings to visualize your specific fluorescent probes. Single Track configurations are used when only one fluorescent probe is present. The list of single tracks is found at the bottom of the Imaging Setup Menu. Multi Track configurations are used when multiple fluorescent probes are present. The list of multi tracks are found under the Acquisition menu button. *In Multi Track configurations, be aware of Combination vs. Sequential. In Combination scanning, the lasers turn on and scan at the same time, and this may lead to bleed through. In Sequential scanning, each laser turns on, scans, and turns off before the next laser fires. Single Track Violet Zen 2009 SOP (06-14) Page 2
INTENSITY ADJUSTMENT Turn off the individual laser lines that you are not adjusting by unchecking the box in the Imaging Setup. Ex: if you are starting with the DAPI laser, turn off the FITC laser. Only the DAPI laser will scan your sample while you are adjusting the intensity, and this will help prevent bleaching of your sample. Select Continuous to find and focus your sample. You can focus your sample by using the focus knobs on the microscope, or the Focus Menu located under the Channels Menu. To adjust the intensity levels of your image, click on the Channels tab, and click on the button of the wavelength channel you want to work on. Pinhole should be set to 1 Airy unit. If you click on the 1 AU button, the pinhole will automatically set to 1 Gain (Master) amplifies the input signal by multiplication, so that bright features are brought closer to saturation and general image brightness is increased. The Detector Gain should be decreased until the saturated pixels are no longer orange. Detector gain should be set to between 500-800 optimally. *Make sure you focus your sample BEFORE you adjust the Master Gain!* Digital Offset sets the gray level of a selected background to zero and adjusts the darkest features in the image to black. To decrease the Amplifier Offset, move the slider to the right. Digital Gain should be left at 1 Turn on the Range Indicator This feature shows oversaturated pixels in RED, while pixels with an intensity value of 0 are BLUE - To get rid of RED pixels, turn down the Gain (Master) - To get rid of BLUE pixels, turn up the Digital Offset Once you finish adjusting the intensities for the first channel, turn off that laser line (Imaging Setup Menu) and turn on the next. Continue in this fashion until all of the channels you are using have been adjusted. Then turn on all of the laser lines and continue. Violet Zen 2009 SOP (06-14) Page 3
SCANNING A QUALITY IMAGE Open the Acquisition Mode Window. check: There are several settings you should Frame size: The larger the frame size, the more pixels per inch. Therefore, as you increase the frame size, you increase the scan time. Speed: adjust based upon your sample. Data depth: 8 Bit gives you 256 shades of grey; 12 Bit gives you 4096 shades of grey. Average: will average the pixel intensity at each pixel based on the number of scans you have selected. You can use the Crop function to zoom in on your region of interest. The Crop button is located on the right menu bar of the image window. When you crop the image, the size of the box outline in Zoom, Rotation, & Offset menu will change to the size/position of your cropped area. When all of your parameters are set, select Snap and your image will start to scan. MODIFYING YOUR IMAGE Once you have scanned your image, you can add a scale bar, add arrows, pseudocolor the channels, or adjust brightness/contrast. The Display button shows your fluorescent intensity range, as well as the controls for Brightness/Contrast. The Overlay button opens a box that gives you the option to add a scale bar, add arrows, or add shapes. Click on the button of the option you want, and use the mouse to place the object where you want on the image. Violet Zen 2009 SOP (06-14) Page 4
SAVING YOUR IMAGE The default save option is (.lsm). If you select the drop down menu, you will have more saving options: Use the Export function in the File Menu to export either the raw data or the Contents of the image window. SHUT DOWN When you are finished, clean the oil off of the objectives (if you used oil) using the Lens Paper, NOT Kimwipes. Open the Laser window and turn all lasers to OFF. Close out of the Zen software. You will get a warning box to leave power on for 5 minutes until the lasers cool. Click OK. While you are waiting, shut down the computer and clean the oil objectives (if they were used). When the fan on the laser unit turns off (it will get noticeably quieter), it is safe to turn off the power. On the Switchbox, turn both switches to the OFF position. (All of the other switches on the system should remain ON.) Cover the microscope. Violet Zen 2009 SOP (06-14) Page 5