Department of Pathology: Confocal Facility

Instructions for Confocal Use:

1. Sign up for time that you wish to use the confocal

2. If this is your initial 5 times using the scope, sign in and have the confocal set-up verified by:

• Kelly Jordan-Sciutto, PhD; room 312; 215-898-4196
• Cagla Akay, MD; room 312; 215-898-4514
• Mike White, PhD; room 312; 215-898-4514

3. Turn on the mercury lamp for the microscope and hit the ignition button. Wait until lamp stabilizes before doing anything else.

4. After the microscope is on, turn on the monitor and computer. Wait until the Login dialog appears and login.

5. Turn on power supply for Radiance control tower (position " | ") and wait for green light to illuminate before proceeding.

6. Turn laser key to the horizontal position and turn on the laser's "bottom up" from the key. Wait for beeping to stop before proceeding. If using the Blue diode, turn the laser Key on the Blue diode box and push the button to turn on the laser.

7. Launch "Laser Sharp 2000" program by double clicking on the desktop icon.

8. Select your login name from pulldown menu and enter your password to begin scanning.

9. To image the microscope must be in the following set-up:
A – the fluorescent filter must be in an OPEN position
B – The bottom right knob must be in the confocal position (position "C")

10. Choose Method
Under the Method pull down menu choose the fluors you plan to use (see fluor chart)

11. Open New Experiment
Pull down FILE menu and click "New Experiment"

12. Choose objective from the Tool Control Panel (Left on Screen, top panel)

13. To Scan click the upper left icon on the tool box (Starburst symbol). Click on the Starburst symbol again to stop the scan.

14. Save images to the hard drive of the computer in your assigned folder. (WARNING: The computers hard drive will be cleaned or extraneous files on a weekly basis.)

15. When finished, contact one of the facility maintenance members (see 2) to check that the set up has been returned to original starting condition. Before turning off the system, please check the sign-up log to see when the next user is scheduled. If there is less than an hour between you and the next user, do not turn off the mercury arc lamp for the microscope. If there is no one scheduled, then proceed to the shut down instructions.

To shut down (after the last user of the day):

1. Log out of the Laser Sharp program.

2. Turn off the lasers from "top to bottom." Turn the key back to the vertical position and turn off the tower (? is in). (NOTE: The fan for the argon laser will run for a few minutes once the laser is off. The fan will automatically turn off once it has cooled down enough.)

3. Shut down the computer and turn off the monitor.

4. Turn off the power supply for the scope. (NOTE: Be sure that the mercury arc lamp has been on at least 1 hour before shutting down. Once this lamp is turned off, it must remain off at least one our before turning on again.)