Short Leica SP8 confocal manual Make sure the objectives are at their highest position and moved out off the way, because the stage will be initialized again TURN ON and Start the SP8 Start Fluorescence source #1 (Leica light source below the table, shutter button have to be pressed) 2 3 4 5 PC Microscope Switch #2, Scanner Power #3 Laser Power #4 and Keyswitch on position ON #5 If you want to use the incubator, turn on the cube on the shelf. If you also want to use CO2, connect the stage insert and adjust the red regulator If you want to use the STED Laser press black button #6 on the laser box, wait until the LED #7 will shine green then orange and again green and only then turn the key#8 to the ON Position The White Laser Line button can always be switched on, so it will start automatically with the system start Start the computer and software Login as LIC User 7 8 6
Operation Life Imaging Center im ZBSA Habsburgerstr. 49 D-79104 Freiburg Starting the software Open the Leica confocal software by double click on the LAS AF icon (it takes a few minutes to boot up) Configuration: machine (used for normal confocal work) or Simulator SP8. Microscope: DMI 6000 (used normally) or manual DM-6 (for image processing - without initialization of the microscope) If you want to use the resonant scanner (for fast live cell imaging) check the box, otherwise make sure it is not checked, can be used with confocal and STED Press OK now the software will initialize the system followed by a query about initializing the motorized stage. This is not necessary unless you plan to do multipoint image acquisition. However, before initialization you must check that nothing is limiting the movement of the stage and you have no sample in the holder. Ignoring this may cause extensive damage to the optics and stage of the microscope! LAS software window open. o At the top of the LAS software window you have a banner for choosing between the main software functionality.
o o o Life Imaging Center im ZBSA Habsburgerstr. 49 D-79104 Freiburg The first slider is for choosing the size of the control icons. This is followed by a drop down menu for choosing between normal confocal mode ( TCS SP8 ) and the various assisted modules (FRAP, FRET etc.). The final element is a four tab arrow (Configuration, Acquire, Process and Quantify). Clicking on any one of these tabs will open a corresponding view in the software Configuration and Turn on the lasers you need: Normally you will only need this tab to turn on and off lasers. Turn on the lasers you need: Configuration tab --> laser Check only the lasers you want to use. Turn on the lasers you need:
Configuration tab --> laser Check only the lasers you want to use. Adjust Ar power (do not use more than 20 %, it is a good start for most imaging) WLL Power typically 70% STED Laser Power typically 80% -100% Aquire: Please note that the scanner is equipped with two hybrid detectors (HyD). These are extremely sensitive. When you use them for the first time you should always check that their gain is set to minimum. If too much light reaches them they will automatically shut down. If this happens it will take a few moments before they recover and you can continue. Change Objectives: It is easiest to change objectives using the software Acquiring Confocal images (Acquire tab, Acquisition sub-tab): Load/save single setting - choose the combination of colours you have in your sample You can adjust the laser power and exact range of wavelengths collected for each PMT Click "live" in the bottom left corner Adjust z-position (=focus), pinhole, zoom Adjust gain and offset for each channel (click the image to select) Choose glow scale (green=under, blue=over) for more accurate adjustments Adjust the line averaging number (marked with red arrow above). You need to empirically determine what is best for your sample but 2-8 are common values.
The number of pixels can be changed from the starting value of 512*512 under "Format". Press Capture for final image, the image window has these buttons (the stack ones are only present if you have a series). Z-stack acqusition Set everything up as above for the brightest region of the stack. Then specify the top and bottom of the stack and the number of slices by moving up and down with the actual plane shown in the z-stack window Press "start" to capture the z-series. It will do whatever averaging you have set for each slice. Sequential acquisition This is worth doing when you have bleedthrough between channels (most common with DAPI and green). Press the "seq" button, which opens the sequential scan box. Click the plus to generate how many scans you need For scan1 set all laser powers to zero except for one laser line, deactivate all but the relevant PMT Scan2, set up the next laser and PMT... Press the "seq" button again to exit sequential mode.
Saving data: Life Imaging Center im ZBSA Habsburgerstr. 49 D-79104 Freiburg Click the "Experiments" sub-tab Save the acquired images for later use: o by saving all the acquired images (Experiment) on the Experiments tab o by exporting images by pressing right mouse button on top of the desired experiment (Right click on each to get various options) Images are stored in leica format (.lif), can export to Tiff (right click on the.lif file to export all at once) If you are using USB memory it is more stable to save to the D:/ drive and then move the data afterwards Press Save All fairly regularly in case of crashes Power down Please clean any oil/water objectives you used and fill out the log book If someone is scheduled within 2 hours - 1. Open the Configuration tab and set AR laser to 0%, turn off any laser that won't be used by the next person (check the booking calendar comments) 2. Click FILE/EXIT to terminate the LAS software. Close all open windows and exit the user profile you are using but do not turn the computer off. 3. Leave the PC, scanner and lasers on. Clean up the objectives; have the lights turned on so that you can see properly. Use lens paper to soak the oil off, but do not rub. If you are the last person of the day or nobody is using the system for at least 2 hours - 1. Open the Configuration tab, set argon laser power to minimum and set all Lasers to the off position. (means: Ar to minimum and all lasers off i.e. uncheck the boxes in config/laser) 2. If you used the STED Laser turn the key#8 to the OFF Position and press black button #6 on the laser box 3. Turn the Laser emission key #5 to the off position. 4. Exit LAS software and turn off the computer. 5. Shut down Fluorescence source #1 6. PC and Microscope Switch #3 7. Scanner Power switch #2 8. Wait at least 10 minutes for the Ar laser cooling fan to turn off, now turn off LASER Power Switch. #4 If you used the incubator and it is appropriate to turn it off (ie you are the last person, or the next person doesn't want to use it), you can turn off the cube on the shelf and the red CO2 regulator at any point. Microscope controls usage The current setting of the microscope can be seen on the info-screen on the frontal side of the microscope
Immediately above there is a simple control panel interface Additional conrols can be found on the sides of the microscope (left and right):
Motorised sample stage Thre is a dedicated SMartMove controller for movement of the sample stage: