Leica TCS SP2 User Manual 1.1 Markus Friedrich and James Lee Department of Biological Sciences Wayne State University Confocal Manual 1
Helpful hints The confocal room is cold. Bring a sweater. Confocaling is like playing an instrument. It takes a while until you consistently generate beauty. Be patient while learning. Otherwise, it may get expensive. Confocal Manual 2
Important rules If you are having a problem don t start playing around. It may get expensive. Call James Lee or Markus Friedrich. Never exchange objectives. This would delete important microscope software settings. If you need to add on additional objectives, let James Lee know. Put your stuff on the counter in the back of the room and not on the microsocope table to prevent misalignment of the potentially vibration sensitive dichroics. Sample Preparation Use of coverslips with 170ul (+/-10um) thickness is recommended. Make sure nail polish sealing has dried for at least one day. Oil immersion: Use Leica oil only! Confocal Manual 3
Operating the Leica DM IRB This is an electronically operated scope. Take the time for making yourself familiar. Otherwise you risk damaging the scope or your samples. The part identification numbers used below refer to the DM IRBE description on page 11 of the Leica DM IRBE manual. Starting up the microscope It is important to start up the microscope before you start up the confocal system. The confocal system needs to communicate with the already running microscope software during startup. You may use the epifluorescence addition on the microscope to examine your samples before confocaling. In this case, you need start up the mercury bulb first (box underneath the microscope table). Note however that it is not necessary to have the mercury bulb running while confocaling. Each mercury bulb costs $300.00. We therefore suggest that you either leave the mercury off or if you need to use it for prior on site evalutation of your specimens turn off the mercury bulb before you start confocaling. Leace the mercury bulb only on if there is a reason to double-check your samples during confocaling. If you turned off the mercury bulb you should wait a minimum of 20 minutes before reigniting it. Start up the scope using the main switch (7) on the left side. The LCD panel on the front of the scope body will first idnicate the initializiation process. One finished you will see a panel shown on the next page: Objective Focus position Focus step lower/upper focus limit Confocal Manual 4
Prizm ring Dry or Immersion Make sure the LCD panel shows the 5x objective in position. If this is not the case change to the 5x by operating the objective wheel which is done using the push buttons on the right side of the instrument. Pushing the lower button gets you to the next lower objective. Pushing the upper button gets you to the objective with the next higher mag. Note: Although stated in the manual the current version of the scope does not discriminate between Immersion and Dry objectives. Note: the objectives are parafocal. That means that each objective will automatically be set at the focus level of the previous objective. You may need to be careful when working with thick specimens and changing to high mag objectives with lower working distance. In this case parafocality may mean squishing your specimen. After positioning the 5x objective bend the upper microscope neck backwards. This opens up plenty of room to mount your sample on the slide holder. Confocal Manual 5
Place your slide with the coverslip facing down. Note again: Never mount slides sealed with finger nail polish where the nail polish is still fresh. Nail polish preparations need a least one day to dry! Left side: Set filter wheel (11) to position 4 Push in UV light block level (behind filter wheel) Right side: Make sure the switch knob on the right side of the transmitted light field diaphragm (27) is set to Vis Turn tube lens module wheel (22) to position 1x Push in path shifter changer, which is close to the tube lens module wheel. Turn the brightness adjustment wheel in your direction to start up the halogen illumination. You should see your sample now looking through the eyepieces. Focus your sample using the either the focussing drive (10) or the push buttons behind the focussing wheel on the left side of the instrument (up is up and down is down). Note: You can regulate the focusing speed of the push buttons by pushing the STEP key on the manual bord. The focussing stepsize is indicated on the LCD screen. S0 = 0.05um S1 = 0.1um S2 = 0.7um S3 = 1.5um Once you located your tissue, switch to 10x objective After switching to 10x mag you should now set an upper focus position. This will minimize the risk that you damage your sample later on by focussing to deep into the tissue. Confocal Manual 6
You can focus by either using the push buttons on the left side of the scope. Upper button moves objective up and thus the focal plane deeper into the tissue. Lower button moves objective down. Focus at the deepest level necessary to investigate you sample. Push the image key for longer than 1 sec. This deletes the previously stored threshold. Push again for 1 sec. This stores your new threshold. Confocal Manual 7
Koehler illumination For optimal illumation of your sample you need to do the Koehler adjustment. Move specimen out of the light path. Close transmitted light field diaphragm (27) by turning holder to the left. Look through the eyepieces and focus the borders of the transmitted light field diaphragm by changing the position of the condenser unit (7 and 8) with the large wheel on the right of it. Center the transmitted light field using the turning knobs on the front of the condenser unit (7 and 8). Open transmitted light field diaphragm (27) until the transmitted light field diaphragm margins disappear from the visual field. Confocal Manual 8
Oil immersion Note: Use Leica oil only! Works best for these objectives. Push the Learn key once to position your objective in 60degree position which makes it best accessible. Add a little aliquot of Leica oil to the lens with the plastic tip, which is linked to the lid of the oil bottle. Be careful not to touch the lens with the plastic tip. Don t use too much oil. It unnecessarily contaminates the objective periphery and increases the tendency of the oil to move away from the lens. Push the Learn key again once. This will bring the objective back to its position underneath the slide. Cleaning the objective after oil immersion use: Note that you do not need to remove the oil completely from the objective lens after you are done. Leica claims that it is not necessary (provided the environment is reasonably dust free). Simply clean off excess oil from the metal periphery of the objective using a clean sheet of lens paper. Water immersion Same as water immersion exept that you simply have to carefully drain off the water from the lens after use. Confocal Manual 9
DIC optics Push in Analyser (21) Chooser correct ICT prism (12) with wheel on the left side. Choice of the suitable objective prism is confirmed on LCD screen. If wrong filter is chosen this value keeps on flickering on the screen. Confocal Manual 10
Switching from DIC to epifluorescence Close transmitted light using brightness adjustment wheel (8). Pull out Analyser (21) Pull out UV light block level (behind filter wheel). You should see your sample after choosing the adequate filter with the fluorescence modul filter wheel. Confocal Manual 11
Getting the scope ready for confocaling Close transmitted light using brightness adjustment wheel (8). Left side: Set filter wheel to position 4 Pull out incident light path switch rod which is behind filter change wheel (11). Make sure Analyzer (21) is pulled out. Right side: Turn right filter wheel to UV confocal lens Pull out path shifter Switch upper left switch from Vis to Scan You are now read to receive signal in the confocal Confocal Manual 12
Starting up the confocal Start up the PC using red switch Log in. Every user has his own account. To start with your last name is username and password. You can change your password whenever you want. Here is an example: Username: Password: family family Push the Scanner switch Push the Laser switch and wait for 30 sec You can now start the lasers you need. Attention: You should only start those lasers, which you actually need for your dyes. If you are not sure about the laser required for your dye see emission spectra table in the appendix (xxx). Argon is for blue dyes (FITC) (458, 475, 488, 514nm and transmitted light channel) HeNe green is for green dyes (TRITC) (543nm) HeNe red is for red dyes (Cy5) (633nm) HeNe Lasers can be simply switched on Argon Laser needs more attention: Make sure that the Argon output knob is at lowest level (full turn counterclockwise). Put key in start position. It will automatically switch back to running position. You are now ready to start the Leica Confocal SoftwareYou are now ready to confocal. Confocal Manual 13
Confocaling This chapter covers the very basics for getting an image for the typical single or multilabelled specimens (GFP, FITC, TRITC, Cy5 or any combination thereof). Any step further may be explored using the Leica User Manual (LUM) or the online help function from within the Leica Confocal Software. Start up Leica Confocal Software by double clicking the xxx file. The welcome window appears. Log in as personal. It takes about 30sec until the program environment appears. At the end of startup we currently get the message DM microscope error in command ( 50013). Code! and the advice to initialize DM IRBE. Press initialize. You will get immediately the message DM IRBE sucessfully initialized. The left monitor shows windows with function menus to operate the confocal, while right monitor shows the Viewer Window, which shows your imaging and image manipulation results (LUM page 48-71). Focus on the left monitor. In the upper panel you will find experiment design functions In the lower panel confocal parameter setting functions The basic operating unit is called experiment Experiment files can contain any number of images from the same experiment, i.e. imaging the same specimens under constant conditions plus accessory files containing the experiment parameters. If you click on NEW a new experiment is started. A new image panel appaers on the right screen. If you close this panel you delete the experiment. The following will lead you through the steps to get a first transmitted light image of your specimen on the screen. All the required function buttons are in the lower half of your visual field. Beam button: A pretty much selfexplanatory Beam Path Setting panel appaers. Choose your wavelenth settings. Activate transmission on the right chooser panel of preconfigured excitation/emission wavelength settings. Our You can leave the panel open or close it. Confocal Manual 14
Unexperienced users are advised to begin with activating TRANSMISSION. You can use this setting to generate a transmission light image of your specimen on the screen to make sure you have your specimen in an adequate optical plane before continuing with activating fluorescent dyes which in the beginning often looks very different from expected. Note the field view is limited at 5, 10 and 20x magnification. From there move to the Signal button: Open this window and leave it open. Lets you modulate contrast (gain) and black value (offset) of the confocal image. This is the main tool to optimize your image contrast. If you activated TRANSMISSION in the Beam button field you will now find that only the PMT Trans is active. Move the Gain level to the bottom. Move on to the Continuous scan button: Triggers continuous scanning. Each new image replaces the previous one. Useful to look through different layers of a thick specimen and for adjusting beam path, pinhole size, electronic zoom and phase (LUM page 36). Sstart confocaling by pushing the continuous scan button. Dim the light now. This will help you to see the image on the screen better. If you don t see your specimen right away, don t worry. Increasing the Gain level for the transmitted light channel (PMT Trans) will help you to visualize your specimen. You will be a little out of focus from the specimen. Use the Z Pos Knob or the microscope focus drive to focus into your specimen. You may have to redefine your upper focussing level on the scope. One you can see your specimen in transmitted light mode you can swith to fluorescent dye excitation settings. Bit depth button Choose between 8 bit and 12 bit gray value. 12 bit is not recognized by some image processing programs. Scan Mode button: Let you choose spatial, temporal and wavelength parameters of your scan (LUM 31-32). Leave at xyz for a start. Confocal Manual 15
Scan Speed button: Fast speed lets you work fast and minimize bleaching but results in background pixels. Slower speed increases signal to background ratio (LUM page 32-33). 800 and 1000 Mh automatically changes to Zoom to 2 and 4 respectively. Start with 400 Mh. Scan Format button: Choose scanning format, which means the number of points that are scanned per length unit along each scanning axis. The lower the format the faster the scan but you might also loose information. Higher formats lead to higher resolution but take longer and may increase bleaching (LUM page 30-31). Start with 256x256. Pinhole button: Let you set the diameter of the detection pinhole. This lets you modulate the thickness of your optical section. Click the airy 1 button to set the detection pinhole diameter to the optimal diameter corresponding to the objective used. Larger pinhole increases signal but also background. Smaller pinhole gives thinner optical section at the cost of signal intensity (LMU page 29). Single Scan button: Triggers one scan. Recommended if imaging bleach sensitive specimens. Confocal Manual 16
Series Scan button: Series Scan button can create an image series, either record serial layers of three dimensional image or generate images of different wavelength one by one. Before recording an image series, configure all required scan parameters using the Continuous Scan function to ensure optimal image quality. To get spatial image stacks: Select xyz or xzy scan mode. Continuously scan and use z pos knob of the control panel to determine the start point. Store the begin point using the Begin button in the Series Scan dialog window. Use the same step to set and store the end point of your image. Select the number of spatial sections using the Sections button. When you choose others, you can define your own desired distance of each layer and the computer will calculate the number of sections automatically. The more sections you take within a specified z-range the more comprehensively you cover you specimen. However, with the number of images also increases the time needed for the experiment, storage space required and memory needed for subsequent manipulation of the images. You therefore will want avoid oversampling. The number of sections needed to continuously cover a z-range depends on many parameters including magnification, wave length and pinhole size. Below are approximate step size values for sampling each section area 2-3times: Magnification 100x 63x 40x 20x 10x step size 200-300nm 300nm 400nm 500nm 1um When you have finished configuring these settings, press the Series Scan button to get the serial images. To view them, you can either press the Gall button in the overview dialog window to show them on the same screen or touch the Play button to see the animated effect. To record a wavelength series: Select a scan mode of either xyλ or xzλ, thus the λscan window will be available now. Confocal Manual 17
Define the wavelength at which you want to begin and save to the scan Begin Point. Define the wavelength at which you want to end and save to the scan End Point. Select the desired number of recordings between the begin and end points of the wavelength series. Open the Sequential Scan order on Beam Path Setting panel. Add your serial wavelength one by one and if you will use them again, save them. Now you are set to get the wavelength series images. Defining user-specific instrument parameter sets: Predefined instrument parameters may not satisfy you. Don t be frustrated. You can have your own settings. On Beam Path Setting panel, you can find the predefined instrument parameters for typical applications. Choose one that is most close to what you want to create and modify the appropriate instrument parameters until you get the satisfied image. Click the Save button to save your new setting into User files. You still can modify your own setting. Just open it, modify it and save again. Other useful buttons: Objective button: Let you switch objectives (see LUM page 25-26) (xxx discrimiates dry vs immersion?) Electronic Zoom button: Let s you zoom into your specimen at increased pixel resolution. Increases bleaching rate. (see LUM page 28) Time button: For setting time lapse series (LUM 34-35). Unidirectional/Bidirectional Scan button: You can double the scanning speed by activating bidirectional scanning (LUM page 45). You may have to adjust phase shift using the phase button. Confocal Manual 18
Phase button: Adjust phase shift when bidirectional scanning (LUM page 46). Scan field rotation button: Rotate scan field. Apply button: Create new experiment taking over settings from previous experiment (LUM 47). Average button: Increase signal background ration using different averaging options (LUM page 48). Saving your data This is covered in the LUM on pages 79-82 Confocal Manual 19
Computer crash Desktop Turn of scanner Wait 10 sec Turn on scanner Wait 30 seconds This may reinitiate your computer. If still unsuccessful, press: contl alt delete Task manager End task or reset button Start program Confocal Manual 20
Shutting down the confocal The most important thing here is the Argon Laser. This Laser must be turned off while the cooling fan is still running. Therefore, take care to proceed step by step. Shut down Leica confocal software Log off system software Wait for message: It is safe now to shut down the computer Switch off computer on knob panel Switch off scanner on knob panel Switch off HeNe Lasers Minimize Argon Laser output on level wheel Make sure the laser fan is still running Turn off Argon Laser using the key Wait 20 min Switch off Argon Laser fan Confocal Manual 21
Shutting down the microscope Clean objectives Set objective in Learn position Excess oil may be removed with cotton swaps but needs not completely removed due to the new Leice formula Severe contaminations: get James Lee or Markus Friedrich. Brush of contamination with Sparkle soaked cotton swap by stroking only once in one direction. Make sure the lowest mag objective is in position in lowest gear Switch of microscope Switch of mercury bulb if used Confocal Manual 22
Important phone numbers Leica 610 321 0460 Brad Calloway (Leica) 1 800 248 0665 x5926 James Lee beeper 313 609 0991 Home 313 273 8004 Markus Friedrich office 7 9612 Lab Home 313 642 0819 Confocal Manual 23
Controling leak through Symultanous vs dual scanning Simultaneous Optimizing AOTF, gain and emission spectrum Saving/calling up parameters dual Optimizing AOTF, gain and emission spectrum Scanning speed difference? Saving/calling up parameterr Effcient data acquisition Good dye combinations Saving files At present the microscope focus is not parafocal with confocal focus which is a little higher. Confocal Manual 24