Setting U P S Equential S Canning ( Recommended) - Leica SP5 confocal User Manual

Setting u p S equential S canning ( Recommended)
1. From the left hand margin of the screen under Acquisition
Mode select the Seq button to open the sequential mode
window
2. Select the + button which will add a scan. Each scan
corresponds to a single laser/detector combination.
Therefore if you are imaging in 2 channels then you will need
to add 2 sequential scans.
3. For a GFP-RFP setup for example you would
select the newly added Scan 1 button then
configure the beam path setup so that only the
488nm laser and the detector assigned to GFP
are active. Use the vertical slide bars to turn all
other laser lines off. Uncheck the other detectors
to inactivate them.
4. Now select the Scan 2 button and repeat the
procedure but this time enable the laser line and
detector for RFP and switch off the first detector
and 488 laser by setting to 0% power.
5. Now when you scan the lasers/detectors are
sequentially turned on and off rapidly to avoid cross talk.
6. Note that there are 3 sequential scan options:
Between lines- (Recommended) The laser currently scanning switches on a line
by line basis. I.e. one line of the GFP channel is imaged, then one line of the
RFP channel etc.
Between frames-An entire image of one channel is created first, then the laser
switches and acquires an image of the second channel.
Between stacks-If acquiring a Z stack, then the stack of Scan 1 (GFP) is
acquired first then the stack of Scan 2 is acquired (RFP).
7. Press the Save button in the sequential scan window and save to your folder on
the D: Disk. Include which wavelengths the sequential scan settings relate too,
to make it easy to select another time.
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