1. Nikon Eclipse TE2000 Spinning Disc

2. inverted perfect focus microscope optimized for live cell imaging and time lapse movies Equipped for DIC, Ludl Piezo Z-stage for 3-D imaging temperature controlled chamber widefield cubes for GFP, YFP, TxRed, CFP, DAPI Cascade 512B and Coolsnap HQ CCD detectors offer simultaneous data acquisition 488 and 568 nm Ar lasers Visitech QLC100 confocal head.

3. Turning on and Setting up the Microscope 1.Turn on Power to the AOTF Controller 2.Turn on Power to the Laser Power Supply *DO NOT TOUCH THE KEY! 3.Turn on Power to the Laser Supply Remote Controller: a: Flip switch to “operate” b: Turn key past “on” and let go c: Laser should go to “100” or more d: Let warm up 30 min before using

4. e: Flip switch to “standby” if not using the lasers for awhile or when you leave f: Laser Power can be displayed in mW or in AMPs depending on which position the toggle is flipped g: Laser Power can be changed by toggling the switch up (increasing power) or down (decreasing power)

5. 4. Open AOTF Controller Software - Adjust % Power in software if needed - Usually start with 100% - Must do this before doing next steps 5.Turn on Black APS Power Supply 6. Turn on Temperature Contoller: - Turn dial to “set” option and use dial to set control temperature. - Keep at 39 o C so that the center of your dish is 37 o C. - Turn dial to T1 to see actual temp. 7.Turn on Ludl Shutter Control: - Widefield/DIC is Halogen bulb - Fluorescence is mercury bulb

6. 8. Turn on Xcite Mercury Lamp 9. Turn on Halogen Lamp *This lamp is under the air table. 10. Turn on confocal head key *Both lights should be on. 11. Turn on the Perfect Focus Box: - Push green button to “engage”. - If green light flashes, it is not working properly. - Be sure to “disengage” PF by pushing green button before removing your dish of cells from the stage.

7. 12. Turn on Power to the Microscope: - Back left bottom of scope where red tape is located. 13. Turn on Camera(s) before opening IPLAB: - Cascade is for Confocal - CoolSnap is for Widefield/DIC/FL 14. Open up IPLAB Software: *You should always get an error message. It is ok. 15. Tell software which camera you’re using: - Select “Camera” option - Select “Select Camera” - Click “OK” by camera

8. 16. Setup camera settings: - Select “Camera” option - Select “Get Camera Info” - Can adjust settings - Gain Index = 3 - Speed Table = 1 - Mult Gain = 3700 17. Adjust Magnification Dial: - Located on bottom right of scope. - “1x” means no additional magnification. 18. Plug in heater controller to stage dish: - Plug temperature probe into small hole on side of setup. - Attach blue clips to each side. - Use the gold ring insert for most MatTek dishes. *Other ring inserts available.

9. Turn off Microscope 1.Turn lasers to zero in AOTF Controller Software. 2.Turn off Power to Perfect Focus box. 3.Flip lasers to “standby”. 4.Turn laser key to “off”. *Listen for fan to get quiet before turning anything else off. 5. Remove sample and clean-up. Liquid waste is nearby and should be emptied if needed. 6. Leave the stage dish in place. 7. Turn off the rest of the equipment once hear fan power decrease. *IPLAB must be closed before camera is turned off. 8.Put cover on microscope. 9.Sign log sheet.