Center for Cell Death, Injury and Regeneration
Before using these instructions make sure you have had at least one training sessions on using the system.
Zeiss LSM 510 NLO with multiphoton excitation operating instructions:
1. Turn on the mercury arc lamp the first if you intend to use it. Wait for 2 minutes for the lamp to warm up before turning on any other piece of equipment. Switch off the arc lamp only after all the other equipment is turned off.
2. Turn key on multiphoton laser box located below the table to the ON position if you intend to use the multiphoton laser.
3. Turn the black stich on the laser box to the ON position. (45 degrees clockwise rotation).
4. Switch on the computer and system component buttons the box besides the computer and switch on the computer.
5. After computer boots up logon with
6. The computer will prompt you to enter your username and password that was created during the training session. This account is used for tracking users and their usage time for billing.
7. Once logged on the computer, launch the LSM 510 Meta software. Start software in Expert mode.
8. Select laser from the 510 control panel and switch on the laser lines that you intend to use and keep the lasers running for 30 minutes before starting an experiment. Do not turn lasers on and off during an experiment. The microscope has the following laser lines available:
A. 458, 477, 488 and 514 nm Argon Laser
B. 543 nm He/Ne laser
C. 633 nm He/Ne laser
D. Multiphoton laser, tunable from 705-980 nm
9. Click on Config and select single or multitrack.Then load the configuration that you intend to use for the experiment using the Config button. Configurations will be created by the facility managers and stored for subsequent use.
10. Select the objective lens that you intend to use for the experiment. Make sure you use the appropriate immersion medium. Do not use the wrong medium as it may damage the lenses. Lenses currently available on microscope:
A. 10X dry lens
B. 25X water immersion lens with correction collar for different coverslip thickness (use distilled water)
C. 63X oil immersion lens (use Zeiss 518F immersion oil)
D. 40X oil immersion lens (use Zeiss 518F immersion oil)
E. 40X dry lens with correction collar for different cover slip thickness
11. Switch to VIS mode on LSM software
12. Mount the specimen on the microscope stage and bring it in focus using transmitted or fluorescent light while looking through the eye-piece.
13. Once specimen is focused switch to LSM mode.
14. Launch the scan menu and select frame size, scan speed and type of scan.
15. Start an experiment with an laser intensity of 0.05%. Use fast scan to bring specimen into fine focus. While doing a fast scan check palette on the iumage window and select range indicator. Looking at the image adjust detector gain, amplifier offset and laser intensity till you get an image with a few red and blue pixels.
16. Switch to slower scan to collect images. Use scan averaging if necessary to improve S/N ratio.
17. Save all your images to the folder you have created under D:\Userdata\Yourname. Before you start saving images you will be asked to create a database. Create the database under the same folder. Also the software saves all images in default LSM format. Use the export function to save them in other formats like TIFF, JPEG. Ask the managers to show you how to use this.
18. Once you finish your experiment shut down all the lasers.
19. Wait for 5 minutes for lasers to cool down.
20. Remove your specimen from the stage and clean the lens using Zeiss cleaning solution while you wait for the lasers to cool down. Also save all your data to a CD, DVD or USB memory. The facility is not responsible for storing images.
21. After the lasers have cooled exit the LSM software shut down the computer and switch the system and components buttons to off.
22. Turn the key on the multiphoton laser to stand by.
23. Turn the black key on the laser box to the off position (45 dgrees anticlockwise)
24. Turn off the arc lamp at the end.
25. Cover the microscope and tidy up the workplace.