MIF ZEISS LSM510 CONFOCAL USER PROTOCOL START-UP Turn on the Mercury Bulb Power Supply (if needed). Power-on the Control Box. Turn on the computer. Open the LSM 510 software. Choose Scan New Images and Start Expert Mode. Open the Laser window, and turn on all lasers. Set the argon laser output to 40%. TOGGLING BETWEEN VISUAL MODE AND LSM MODE You control the objective and the reflector wavelength using the touch panel on the right of the microscope. Please note that the 63X objective is a WATER lens. Do not put oil on the 63X objective! In order to change between the Visual and the LSM mode, use the slider rods on the lower right of the microscope. The button on the LSM menu bar will not work on this system. Side Port When the top slider is out, and the bottom slider is in, the microscope is LSM in LSM mode. (Reflector must be on Slider Rods Open ) TV When the top slider is in and the bottom slider is out, the microscope VIS is in Visual mode. In this mode you can look through the eyepieces and change the wavelength of light using the buttons on the touch panel. All of the objectives are parfocal, so you can now use the objective buttons to switch between objectives. Remember to clean your slide if you move from an oil lens back to a dry lens! LSM510 Confocal User Protocol SOP (12-12) Page 1
FLUORESCENT PROBE CONFIGURATION The Configuration Control window 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. Multi Track configurations are used when multiple fluorescent probes are present. 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. Choose either Single or Multi Track, and click on the Config Button on the right of the Configuration Screen. A drop down box will appear. Scroll through the list to find the correct set of configurations that match your specific fluorescent probes. Click Apply, and close the drop down box. INTENSITY ADJUSTMENT Switch from the Vis setting to the LSM using the Side port sliders. Open up the Scan Window. Run a Fast XY scan to find and focus your sample. You can focus your sample by using the focus knobs on the microscope, or the Stage Control box located on the LSM browser menu. Once the sample is in focus, click on the palette button on the image window. Choose the Range Indicator option. This will show you areas of your image that are either too bright (orange), or too dark (blue). LSM510 Confocal User Protocol SOP (12-12) Page 2
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 button, the pinhole will automatically set to 1 Detector Gain 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. Amplifier 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. Amplified Gain should be left at 1 SCANNING A QUALITY IMAGE In the Scan Control Window, select the Mode button at the top. There are several settings you should check: Frame size. The larger the frame size, the more pixels per inch. Therefore, as you increase the frame size, you increase the scan time. Scan speed: Normally start at 6 and adjust based upon your sample. Data depth: 8 Bit gives you 256 shades of grey; 12 Bit gives you 4026 shades of grey. Average: will take the average of 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. LSM510 Confocal User Protocol SOP (12-12) Page 3
When all of your parameters are set, select Single 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 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. The Chan button allows you to change the color of each channel in your image. The Contr button allows you to change the brightness and contrast of the image. SAVING YOUR IMAGE All images are saved as.mdb. In the LSM Browser, select the File button. Go to Save As Create new MDB file. Place your folder in the Users Directory, and name it so you can keep track of which images you have taken To convert an image into a TIFF, click on Export. You can save your file in two ways. If you select Raw data single plane, all of the information about that image will be saved. If you made any changes in the contrast or brightness, it will not carry over. You are saving the Raw Image file. This is the most quantitative way to save your data If you select Save Contents of the Image Window, you will get the image that you have modified. When you are finished, clean the oil off of the objectives using the Lens Paper, NOT Kimwipes. LSM510 Confocal User Protocol SOP (12-12) Page 4
SHUT DOWN Open the Laser window and turn all lasers to OFF. Close out of the LSM510 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). Turn off the Remote Control Box. Turn off the Mercury Bulb Power Supply. Cover the microscope. LSM510 Confocal User Protocol SOP (12-12) Page 5