Olympus FV1200 User guide
June 23, 2015
6.
Set up proper acquisition condition each fluorescence colors by adjusting laser power, PMT detector
sensitivity, and offset. The following procedure is for Alexa Fluor 405, EYFP, and Alexa 633 as an example.
a) Check the Laser 405 only (check off other lasers, i.e. 515 and 635) to set up the imaging condition for
Alexa 405.
b) Click
and focus onto the brightest focal plane of your
sample.
c) Press
Ctrl-H
keys to change the color LUT to Hi-Lo; the live display
will change to blue/white/red color scheme, where red pixels
represent saturated intensity (beyond scale), blue ones complete darkness (0 intensity),
and white in between.
d) Adjust the laser power (at least 5%) and HV and Offset of CHS1 to obtain some
brightest pixels in red and the background in blue.
e) Click
to stop the fast scanning and click
to scan at the imaging speed
and to check the image quality. Adjust more if needed.
7.
Repeat the above procedure for 515 nm (EYFP) and for 635 nm (Alexa fluor 633).
8.
Check on all the selected lasers.
9.
Set the Zoom and Rotation as required.
10.
Click
button to capture images. When acquisition scanning is done, a 2D view window will
appear.
11.
Save the image in .oib or .oif format. (.oib format is more convenient.)
(Optional Spectral Setting for Multicolor Imaging).
Often excitation/emission profiles of fluorophores in multicolor imaging
may be close, so there could be possible bleed-through of a fluorophore
emission to neighboring channels. To minimize this artifact, the emission
detection range of each channel can be adjusted by changing
Spectral
Setting
.
Click on
, it will bring
Spectral Setting
window. Change the range of
spectrum for each channel (CHS1 and CHS2) by sliding, widening, or
narrowing the tabs
or arrowheads.
