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Biological Imaging Section

Protocols: Leica SP2-AOBS Confocal Microscope

Startup Procedure

  1. If you are going to use the UV laser, this must be turned on first to prevent any possible damage to other pieces of equipment. To turn on the laser: Flip circuit breaker on the power supply to the on position and allow a few moments for the cooling system to start. Turn key to on position and wait for fan to start. After a few minutes a squeal will be heard—that is the ignition of the UV laser. Now it is safe to proceed to the next step. Be sure the power level control is 1/4 turn clockwise above 0 such that the current limit light is off after ignition. This level will need to be increased before scanning by rotating the power level control knob .5 turns clockwise (to a 1 or 2 o'clock position).
  2. Switch on epifluorescence lamp power supply (beneath microscope table on left).
  3. Switch on laser. The 488nm laser (FITC) is controlled by the leftmost switch on the power control box. First switch on the power and cooling fans by moving the red switch to the on position. Wait a minute or so, then turn the key to the on position. The key must be turned momentarily beyond on to start the laser. Switch on the Ar laser if you need 458nm, 488nm, or 514nm, or you need to collect DIC images.
  4. Switch on microscope. The switch is on the left side near the front.
  5. Switch on the PC with the rightmost red switch on the power control box. Wait until the Windows login box appears before proceeding. Login to the confocal computer with the domain set to Leica SP2-424. After login, wait 1 minute to be sure the virus scan software is finished loading.
  6. Switch on the scan control electronics. This is the center red switch on the power control box. The scanner will take approximately 1 minute to initialize; please wait before proceeding.
  7. If you need 543nm (Cy3), 594nm (TR), or 633nm excitation (CY5), switch on the HeNe lasers. These are the upper center and upper right keys on the power control box. The upper center key is for the 543 and 594 lasers, while the upper right key is for the 633nm laser. Only turn on the lasers you need.
  8. Double click the Leica icon to load the confocal software. This will take a minute to initialize. If you want to use the software for image analysis only, with no acquisition, do not use the icon. Go to Start --> Programs --> Leica Confocal Software --> Simulator. This will give you software access without switching on the electronics (Note: choose Leica confocal software, fifth from bottom, not the Leica confocal software with the red and yellow icon).
  9. The Leica software will come up with a dialog box listing your name. Click start to begin the initialization. This will take approximately 1 minute.
  10. Once the main menu has opened, click beam to bring up the detector/laser control dialog box. Select a method from the Leica list or from the user list at the bottom. Double-click the method to load. Selecting a method will bring up all the parameters saved with the method. It will not change the objective, so you must check this either in the software or on the microscope.
  11. At the lower right of the menu monitor, click the folder button and select a control panel template. Normally the one to select is Control Panel-Username.
  12. Be sure to click lens and check that the objective is in position before beginning to scan. This is especially important if UV excitation is to be used. The microscope turret will automatically move to the correct position once this information is entered.
  13. Click on the frame average button and be sure to enter 4 (the default is 1). A setting of 4 is good for most frame averaging operations.
  14. A scan speed of 400Hz is recommended for most normal operations. This is the default setting.
  15. Click pinhole --> airy 1 to select the standard pinhole for the objective you have chosen.
  16. If you are using UV excitation, click UV lens to confirm that the appropriate UV compensation lens is selected. This should match the objective you have selected.
  17. Select mode and choose the appropriate mode for your experiment. If you are doing time series work, xyt or xyzt must be selected, otherwise the time functions will be grayed out.
  18. Adjust the AOTF sliders to get reasonable brightness using the least amount of power. To protect your samples, it is far better to use higher PMT gain (700–900 v) than to increase laser power. Typical AOTF settings are Ar 15%, Orange HeNe 30%, Red HeNe 15%. The UV laser is limited to the discreet steps of 12.5%, 25%, 50% and 100% transmission. Normally 12.5% is used. Minimization of cross talk should also be considered when setting laser power.

File Saving Procedures

The confocal software saves files in a database system, with a file extension .lei. There is also a text file saved to allow you to view the file parameters from outside the Leica software.

  1. Files can only be saved from single scan or series modes. "Continuous" is only for viewing the image on screen and setting the parameters. Once the parameters are set, click single scan to collect a frame averaged image.
  2. Click on the image name, Image XX, which appears in the database window. The name should become highlighted, and a box will enclose the file name. You can then type a new file name. If the name does not change to a dialog box, click a second time. (If you prefer, you can follow instructions 3 and 4 below.)
  3. Right click on the highlighted image name, then Rename. A dialog box will appear around the image name. Enter the new name then enter.
  4. After the first image has been collected and named, the database can now be named and saved. Highlight a file, or the database name, then click Save in the buttons above the database window. You can now name the confocal session (folder) (called an experiment by Leica) with either a date or another name. Be sure that the location for saving is correct. The experiment should be saved in D:\users\username\experiment. Be sure it is not being saved in C:\temp (a system default for the first use).
    The blank in the dialog box is labeled file name, but is in actual fact the name of the folder you are creating for the sessions experiment. You do not need to create a new folder manually, this will be done by the software when you press Enter. Note: you are naming the file called experiment.lei, not the line above called "experiments."
  5. Once you have named the database file you can resume collecting images. Continue to name your files by clicking and highlighting or naming through Rename. WARNING: THESE NAMED FILES ARE NOT ACTUALLY SAVED. They are stored in RAM until you save them. Should the system crash, all of these files will be lost. Therefore you should click save every few files, to ensure that the data is written to the hard drive.
  6. Files saved in the Leica database system are directly readable in Imaris or in Adobe Photoshop. There is no longer a need to use file export. However, each channel of your images will be saved as a separate file. Imaris will re-associate these separate channels.
  7. File movements: Although you are logged into the local machine, and not the network, there is no need for a new login to transfer data to your area.
    Go to Start --> Run, type \\Niaid-file1\Username$ --> Enter. The next dialog box will request your username and password. Be sure you enter your username as "niaid\username," and your normal network password, then Enter. You will now get a box showing your network directory. You should also open "my computer," D --> Users --> Username, and then copy paste from one to the other.

Shutdown Procedure

  1. UV laser: On the UV laser, turn the knob counterclockwise but not all the way down. Be sure that the current limit light does not come on (if it does, you have gone too far). Allow the laser to run at this level for 3 minutes, then turn off the key switch.
  2. Visible lasers: Turn down the Ar laser power with the black power control knob. For the Ar laser turn the knob fully counterclockwise. Allow the laser to run at low power for 3 minutes, then turn of the key switch (counterclockwise). For the CY3, TR or Cy5 (633) lasers just turn off the keys (These are the upper rightmost and center keys on the control panel).
  3. Allow the lasers (including UV) to cool for 10 minutes with the key off, but the fan running, before switching off the red power switches. THIS STEP IS CRITICAL! (During this 10 minute cooldown period, you can proceed with the rest of the shutdown procedure.)
  4. Exit the Leica software.
  5. Turn off the Hg lamp (epifluorescence) with the switch on the white box on the floor on the left side of the microscope table.
  6. Turn off the scan control electronics (center red switch on the control panel).
  7. Turn off the microscope (left side at bottom). Before doing so, you might want to lower the objective turret.
  8. Exit Windows XP (start --> shutdown --> shut down computer).
  9. The computer will shut down automatically. Next turn off the switch labeled PC on the control panel. This switch will turn off the two monitors.
  10. Turn off the power to the laser: For the Ar laser switch off the left red switch on the control panel.
  11. Shut off N2 to floating table (in hallway, middle tank). Do not adjust the regulator pressure. Close the tank valve on the N2 tank (counterclockwise).

Correct Use of the DMIRBE Microscope Program

The Leica DMIRBE microscope uses its own internal program to control the objective turret and focus motor. All of the objectives, Wollaston prisms, and phase rings have been programmed in already. Please do not change these settings.

All of the objectives to be used in the confocal imaging mode are oil immersion objectives and have short working distances. Place a small drop of oil on the objective before putting your slide on the stage. Different brands of immersion oil are not miscible and will cause serious imaging problems if mixed. If in doubt, be sure and clean your slides of any old immersion oil before starting.

The basic controls you will need to use the microscope are as follows:
  1. The two buttons on the right hand side of the microscope control the movement of the objective turret up and down. When pressed simultaneously, they switch the focus motor to the coarse focus function.
  2. The two buttons on the left hand side of the microscope control the choice of objective. The upper button moves to the next highest power objective, the lower button to the next lowest. If either of the buttons is depressed longer, an objective can be skipped in either direction. If you have not set the upper and lower limits for the objective turret, DO NOT PRESS EITHER BUTTON! This can cause the turret to rotate without lowering the objective.
  3. Setting the upper and lower turret movement limits: Place a slide on the stage—coverslip side down. Lower the objective turret 3 – 4 mm below the slide using the lower button on the right side. Press the lower limit button on the front of the microscope and hold it until "del?" appears. Release the button, and press again and hold until "Set?" appears. Release the button. A should appear in the microscope control window now. Slowly raise the objective using the upper button on the right-hand side of the microscope until the immersion oil just makes contact with the slide. Now gently raise the objective using the focus knob until the sample comes into focus. Press the upper limit set button on the front of the microscope and hold until "Del?" appears. Release the button. Press and hold the button once again until "Set?" appears, then release. A symbol should appear in the microscope control window, and the display should say "0um." This will set the upper limit for the raise/lower controls.
  4. With the exception of the 10x and 20x objectives, all require immersion oil. In order to put oil onto the objective, you can rotate the objective turret gently by hand or use the "learn" button. Pressing the learn button once will cause the objective turret to rotate and present the objective you were using at the rightmost position. Apply the immersion oil to the objective, press "learn" once again, and the objective will return to its initial position.
To switch between epifluorescence and confocal scanning:
  1. Close the epifluorescence shutter with the slider on the left hand side of the microscope.
  2. Rotate epifluorescence filter wheel to scan position.
  3. Rotate magnification wheel to scan/UV.
  4. Pull out slider to open confocal port.
  5. If necessary, switch brightfield light source control from view to scan.
  6. Be sure the Wollaston prism wheel is set to blank position. The DIC prisms will cause the confocal fluorescence images to be much less sharp.
To collect DIC images through the confocal:
  1. Be sure to have the illumination control knob switched to scan.
  2. Rotate the Wollaston prism wheel to the appropriate position for the objective you are using. This will be indicated in the window on the front of the microscope. Match the letter in the second position to the letter specified by the objective.
  3. Be sure the Wollaston prism selected on the condenser filter wheel is the correct one for the objective you are using.
  4. On the main control window, turn on the transmitted light detector.
  5. Switch one of the buttons on the panel box to PMTT (PMT transmitted). (I suggest using the pinhole control knob because this on is seldom used.)
  6. Turn off the fluorescence detectors so that the images collected will be single channel.
  7. While scanning, move the Wollaston prism slightly to vary the amount of "shear" in the image of the specimen.
  8. Adjust the "PMTT" and "Black level" controls to yield a nice image.
  9. Save the image in the usual manner, and the DIC image will be saved as an additional channel. You can also click on the DIC image, then right click send to Experiment --> snapshot. This will save the DIC image as a separate image.
Saving the overlay image for later viewing in Photoshop:
  1. Open the confocal file.
  2. Select tiled.
  3. Select overlay. Note there are two forms of overlay, an RGB format and a true color. Choose the form you want.
  4. If the file is a z series, project the file or select the appropriate plane of section.
  5. Right click inside the overlay image and select send to --> experiment --> raw.
  6. A file will appear in the database window as "Overlay000." The file size should be 769K (24 bit color file).
  7. Right click the filename Rename and then rename the file to match the original data.
Note for Adobe Photoshop users:

If you are planning to open and adjust your images in Adobe Photoshop, the following initial procedures are recommended:

  1. Click Image --> Mode and be sure it is set to RGB color.
  2. Click Image --> Image size and set the resolution to 300 dpi. Before doing this, be sure the resample box is ticked, as well as the constrain proportions box.
  3. Set image size to 4 x 4 (or 5 x 5) to reduce the file sizes slightly.

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Last Updated April 05, 2007