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Rolly Wiegand – CALM Leica SP5 manual
15
Fig. 18
Control console
Fig. 19
display mode, pixels that are close to saturation and over appear blue (see Fig. 21, right
window for channel 2), whereas pixels that turn green indicate a too low off-set (see Fig.
20, window for channel 1, left panel). Start continuous scanning by clicking the ‘Live’
button at the bottom of the main control panel. Adjust the off-set by slowly turning down
the ‘Off-set’ button on the control console (see Fig. 18) so that the percentage becomes
more negative until the first green pixels appear in the displayed, scanned image. A typical
setting would be -0.4%. Check whether this setting is accurate for optical sections
throughout your sample by slowly moving the Z-positioner on the control console up and
down (see Fig. 18), and adjust if necessary. Once you have finished optimising the PMT
gain (see 4.4.5.2.), check the off-set settings again and correct if necessary.
4.4.5.2. Setting the PMT gain
Whilst continuing to scan, adjust the ‘voltage/gain’ setting on the control console (the
range is 0 - 1250 V and the default 0 V) until the first pixels turn blue (see Fig. 21,
channel 2). Turn the gain slightly down a bit until no more blue pixels are visible in the
display panel and check this setting for other optical sections in the sample. A typical
setting would be between 600 and 1000 V. Avoid too high PMT gain settings because this
causes a significant increase in noise in your image. Once you have reached the
maximum voltage possible (1250 V), this will no further increase if you turn the knob on the
control console, and this will be indicated by a beeping acoustic signal.
If both settings are optimised, pixels across the image and different optical section should
be in the range of glow tones from dark red to bright yellow and white, but no blue and only
sporadic green pixels should be visible (see Fig. 22). Once you have adjusted the PMT
settings for one channel, proceed with adjusting all other channels in the same way.
Gain
Offset
Z-position
