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Setup for epi-illumination to view through the eyepieces:
1. Choose dichroic for appropriate fluorophore.
2. Open shutter at epi source and shutter on front of scope (move to 'O' from 'C').
3. Turn off brightfield.
4. View sample through the eyepieces.
Setup for confocal imaging:
1. Choose position '6' on the filter wheel.
2. Close the shutter on the front of the scope (move to 'C').
3. Turn off brightfield.
4. Choose the Objective that you are using in the top right box (this is very
important so that you get the correct scaling!).
5. Turn on and off the detectors and lasers that you want to use (remember that
they are on when the background is colored, and they are off when the
background is black)
a. If you are using 'frame lambda' set up each pass individually
b. Set your pinhole: generally it is best to leave it at a small (30 micron)
pinhole unless there is a compelling reason to change it
6. If using the standard detector set the detector gains at 6.5 (your usable range is
generally between 5 and 8)
7. Go live and slowly turn up the laser power to your sample and then
increase/decrease detector gain as needed. Typical laser powers: <20% for
408nm, <30% for 488nm, <50% for 561nm.
a. It is important not to over saturate your image, you can check this by
making saturated pixels colored
i. Go to Color and click the check box 'add saturation indicator'.
b. Do this for each frame lambda pass
c. When finished setting up your passes uncheck the 'preview pass'
checkbox to allow you to take all passes at the same time
8. Average:
a. Use this to decrease noise in your sample without increasing laser power.
b. In order to use this you need to press on the green check box on the top
window
9. Z-stack:
a. Clear everything by pressing the red bars.
Filters for the Eyes
Fluorophore Cube
D/F/R
1. D/F/R
DAPI
2. UV2E/C
340-380
Ex
Em 435-485
CFP
3. CyGFP
GFP
4. B2EC
Ex 465-495
Em
Rhodamine
5. G2E/C
Confocal
6. Empty
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