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Rolly Wiegand – CALM Leica SP5 manual
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Thumb rules for adjusting the slits:
The slits should be set so they do not overlap with any of the laser lines.
The slit opening should be as close as possible to the emission maximum of the
fluorophore to be imaged.
The narrower the slit the more specific and less prone for cross-talk the setting is.
However, if the signal from a dim label is too weak to detect, the slit may be opened in
order to collect enough photons. The user has to optimise the balance between specificity
and the need to collect sufficient emitted photons.
The settings of the slits will be recorded with the image data as metadata and can be
retrieved at any time after acquisition. However, it is recommended to take notes of the
exact settings.
The settings of the slits can also be saved with the other beam path settings (see 4.4.7.
and Fig. 15 F).
Fig. 17
4.4.5. Adjusting the PMT settings
The CALM SP5 system has 3 different PMTs unlike the schematic representation in
Fig. 15. To use a PMT it must be activated by clicking the appropriate tick box (Fig. 17). By
clicking the individual ‘PMT’ buttons, a little window pulls out, which allows to set the gain
and the off-set of the individual PMT. These two parameters can also be set by using the
control console (see Fig. 18). The accurate adjustment of each PMT with regard to each
fluorophore signal is absolutely crucial for a valid image acquisition. For instance, gain
settings that lead to saturation and thus clipping of the intensity information in the image,
render the resulting data useless for accurate quantitation. These settings determine how
optimal the system uses the whole dynamic range of the digital image. The off-set defines
the intensity of the ‘darkest’ pixel in the image and should be set so that it is just above ‘0’.
Fig. 19 A shows the intensity frequency histogram of an image with too low settings for
both, the off-set and the gain, whereas panel B shows the situation with both settings too
high. Both inaccurate settings will lead to a poor use of the dynamic range and to clipping
of the intensity information in the image. Fig. 19 C on the other hand shows an intensity
frequency histogram with optimal setting of PMT off-set.
4.4.5.1. Setting the PMT off-set
The easiest way to adjust the PMT settings is to focus on a middle section of your
specimen with high intensity features. Click in the display window (right monitor) on the
panel representing the first spectral channel to activate the control panel for this channel.
The activated channel is indicated by a dotted frame around the respective image panel,
e.g. channel 1 in Fig. 20. Then change the look-up table (LUT) to glow scale (GloOU) by
clicking the button indicated by the circle and arrow in Fig. 20. By clicking this button
repeatedly, it will change through LUT-GloOU-grey scale, consecutively. In the GloOU
