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Rolly Wiegand – CALM Leica SP5 manual
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4.4. Generating new Beam Path settings
The Beam Path window (in the acquisition tab) is a schematic representation of the major
components of the scanning system (see Fig. 14). It shows the laser modules with all
available excitation lines on the system (Fig. 14 A), the objective lens selector (B), a
spectral view help tool (C) and the module for the detector (PMT) settings (D). To create
new settings, you can start from scratch or use predefined settings and modify them (see
4.7.7. for loading predefined settings).
4.4.1. Excitation laser line selection
Switch on the required laser source (UV and visible) by clicking the appropriate grey dots
in the laser control window (Fig. 14, A). If the dot turns red, the laser source is activated. In
the same window, set the sliders of all laser lines you want to use to the percentage of
laser output you require. It is recommended to always start with as little laser power as
possible to avoid bleaching and photo-toxic effects. Typical ‘start settings’ for well labelled
specimens for the different laser lines are as follows:
405 blue diode – 20%
Argon laser – 5-10%
All Helium Neon lasers – 50%
As a visual help, the grey lines representing the different paths of the laser lines in the
‘Beam Path Settings’ window turn coloured when the appropriate laser line is activated.
Should the laser power not be sufficient, it can be adjusted any time. Try also to choose
the laser line as close as possible to the absorption maximum of the given fluorophore.
4.4.2. Spectrum view tool
In order to assist and facilitate the definition of the beam path settings, the software
provides a visual tool that shows the excitation lines as well as the emission spectra of a
large range of commonly used fluorophores. To use the tool, one emission spectrum can
be chosen for each channel by clicking the fluorophore setting in each PMT box (Fig.
14 E). This pulls out a list with fluorophores you can choose from. The emission spectrum
will then appear in the Spectrum View Tool as a dotted line, accordingly (Fig. 14 C).
Note: This is just an auxiliary tool that has no impact on the actual settings.
4.4.3. Spectroscopic separation and detection of the emitted fluorescence signal
The Leica SP5 uses a sophisticated filter-free technology to separate the emitted light from
the fluorescent sample into different spectral bands. In brief, the entire emitted signal is
spectrally dispersed by passing the light through a prism (see Fig. 15 - 1).
The spectrally dispersed signal is then passing through a lens that projects the light
parallel onto the first slit in front of the central PMT. The slits are adjustable in width and
position, and the mobile elements (sliders, see Fig. 15 – 2) that generate the slit have
highly reflective surfaces. Depending on the opening width of the slits a certain spectral
band of the signal can pass through it and is collected by one of the PMTs. The light of
shorter or longer wavelengths is reflected by the sliders onto the next set of slider
elements, which in turn can be adjusted to define different spectral bands for detection in
separate PMTs. The lack of filters in this system provides a superior technology with
respect to sharp spectral separation and guarantees high transmission properties,
something a filter-based system cannot deliver.
