Follow text file SOP for 750 laser.
Aligning back optics
- place microscope in epi mode (12.000)
- Course tuning
- remove 20x, 100x objectives.
- screw in target objective in 100x objective position.
- remove final lens before scope body, add iris in place of back lens
- tighten iris at end of columnating beam (mounts the different lens)
- focused beam should center on back iris tightened and on target.
- Walk beam using two guiding mirrors. First mirror is for centering on first iris, second mirror for centering on objective target (can also use roof target). Generally best to overshoot and correct back (since moving second mirror will misalign first).
- Finer tuning alignment
- add back objectives. Place coverslip on lens. center focus (can use focus lock as guide)
- Open backport iris (avoid extra diffraction affects).
- Place index card squarely over objective. Should get a good diffraction pattern on the index card.
- Turn on camera. Might see diffraction pattern (clearer with 750?). Really only need center spot. Can do this in quadview mode. Use Grid or target to see center of dot. Make this centered (use two walking mirrors). (should simultaneously center for all quadview fields if in quadview mode).
- Make sure both dot is centered and that diffraction pattern is symmetric (also involves gently walking of mirror).
- replace final lens before scope body. Height should be just above the bottom, otherwise insufficient dynamic range to correct position with tweaking screws. should be straight. Tune side and top tweaking screw so that beam centers on spot.
- System should be centered.
- Bead check
- use bead field to check flatness of field.
- if individual color channels are out of align, might be fixed by tuning channel specific dichroic mirrors.
- check TIRF. Remember TIRF different for different colors, can lead to flatness of field effects if on edge of being past TIRF.