(only 10x and 20x are installed on Microscope; others are in cabinet)
5. Photobleaching / photactivation cube (has an OD2 excitation filter and a TB 355-405-473 dichroic mirror)
|Date||405 nm||488 nm||561 nm||640 nm||Notes|
Open the Projector plugin in Micro-mananger to control the photobleaching system. Instructions on using the Projector plugin are here. To control the laser powers, open the Vortran control panel. To calibrate either laser, you can use an 0.01 mg/ml fluorescein solution, imaged in the FITC channel. A bottle of this solution is in the cabinet. Right now there is no way to save the calibration, so if you switch lasers you'll need to recalibrate.
For photoconverting mEos2, using the correct laser power is critical. If the laser power is too high, the protein will bleach instead of photoconverting. With the current OD2 filter, a 405 nm power of 50 mW works well (once we switch to an OD1 filter, use 5 mW). The Rapp unit ignores the spot dwell time parameter; to control how long the area is converted, adjust the loop parameter. For the laser power above, 10 loops gives good photoconversion and is still relatively rapid (~1s for areas of a few μm2 at 100x). For bleaching GFP you'll need to use higher power.
Laser powers measured out of 10x objective, each laser at 50 mW power (no ND filter in)
|Date||405 nm||473 nm|
|3/17/2014||10.3 mW||9.0 mW|