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Rolly Wiegand – CALM Leica SP5 manual
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The example in Fig. 13 shows the excitation (dotted lines) and emission spectra (solid
lines) of 3 different fluorophores. The arrows display the 3 laser lines used for excitation.
The bars below the spectra indicate the 3 spectral bands separately collected by the
detectors. In this example, three possible scanning modes can be used:
1 – simultaneous scanning of all 3 channels – Cross-talk is a problem, because for
instance part of the signal from the second fluorophore will be recorded in channel 3
2 – sequential (all 3 channels separately) – Cross-talk is ruled out by sequential scanning,
the drawback is the scan time increases three-fold
3 – sequential/combined (channel 1 and 3 in the first and channel 2 in the second
sequential scan) – This combined method involves 2 sequential scans. During the first
scan channels 1 and 3 are recorded. Cross-talk is ruled out because both excitation and
emission spectra are far enough apart and thus do not affect the recording of the opposite
channel. In the second scan, the second fluorophore will be excited with the 543 nm line
and separately recorded. This method rules out cross-talk problems and saves some time
compared to scan method 2 (about 30% shorter scan time). It is also recommended to
combine the acquisition of the transmitted light channel with the channel that uses the 488
nm Argon laser line (see also 4.4.6.).
Section 4.5. explains how to set up a sequential scan.
A = Laser control module
B = Objective selection tool
C = Spectral view tool
D = Slit settings
E = PMT settings
F = Control for
saving/loading of settings
Fig. 14
‘Beam Path Settings’ windows
B
C
D
A
F
E
