A femptosecond pulsed laser is used for two or three photon excitation of fluorophores. Especially for thick and live material, this has benefits over standard confocal microscopy. A more detailed explanation of the MP microscope and why we purchased it can be found here.
Sign-Up in advance
The multi-photon attachment can be used on either an upright or an inverted microscope. It is important to know before signing up which microscope the multi-photon attachment will be on. To minimize switching the microscope back and forth, we encourage users to coordinate their experiments with other users and to book blocks of days with breaks for other users.
All appointments must be approved before sign-up by Michael Cammer or Jeff Wyckoff. The laser may be tuned by Jeff Wyckoff (x4113), Michael Cammer (x2890) or designated Dept. ASB Biophotonics Innovation Lab people only. No users are allowed behind the floatation table.
Training for unassisted microscope use is done on the standard BioRad Radiance 2000 confocal microscope.
Instructions
The air conditioner and the laser cooling unit must be on.
The room must be cool.
If there is a loud beeping or wail coming through the wall, then the
laser cooling unit is off.
Mercury lamp must be turned on before other components. Do not turn on Hg arc lamp if laser or scan unit is on!
If using 960nm excitation (e.g. for GFP or FITC), ultra-purified N2 purge must be turned on a minimum of 2 to 3 hours before use of instrument.
Laser must be turned on to warm up one hour before use.
Power strip to far left turns on oscilliscope, Reese and detector for monitoring laser.
Computer must be fully on and booted up before turning on scan unit.
Software is the same LaserSharp software as normal confocal.
Lights must be off in room while imaging.
Simple GFP method | |
Filter 1 | green on screen is blue emission red on screen is green emission |
Filter3 | green on screen is green emission red on screen is red emission |
Filter wheel set for EXT/DDS for multi-photon imaging.
Tricks for tuning laser at 960 nm.
Shutdown checklist
This is a checklist. Please run down each point every time you are done working.